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Site Specific Microbiome of Leishmania Parasite and its Cross-talk with Immune Milieu


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Microbiota consists of commensal, symbiotic and pathogenic microorganisms found in all multicellular organisms. These micro-organisms are found in or on many parts of the body, including the intestinal tract, skin, mouth, and the reproductive tract. This review focuses on interplay of site specific microbiota, vector microbiota along with immune response and severity of Leishmaniasis. Herein, we have reviewed and summarized the counter effect of microbiome post infection with the Leishmania parasite. We have studied skin microbiome along with the gut microbiome of sand-fly which is the vector for transmission of this disease. Our major focus was to understand the skin and gut microbiome during Leishmania infection,their interaction and effect on immunological responses generated during the infection.Moreover, systems biology approach is envisioned to enumerate bacterial species in skin microbiota and Phlebotmus gut microbiota during Leishmania infection.
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Immunology Letters
journal homepage:
Site specic microbiome of Leishmania parasite and its cross-talk with
immune milieu
Pragya Misra, Shailza Singh*
National Centre for Cell Science, NCCS Complex, Ganeshkhind, SP Pune University Campus, Pune 411007, India
Immune system
Gut microbiota
Microbiota consists of commensal, symbiotic and pathogenic microorganisms found in all multicellular organ-
isms. These micro-organisms are found in or on many parts of the body, including the intestinal tract, skin,
mouth, and the reproductive tract. This review focuses on interplay of site specic microbiota, vector microbiota
along with immune response and severity of Leishmaniasis. Herein, we have reviewed and summarized the
counter eect of microbiome post infection with the Leishmania parasite. We have studied skin microbiome
along with the gut microbiome of sand-y which is the vector for transmission of this disease. Our major focus
was to understand the skin and gut microbiome during Leishmania infection,their interaction and eect on
immunological responses generated during the infection.Moreover, systems biology approach is envisioned to
enumerate bacterial species in skin microbiota and Phlebotmus gut microbiota during Leishmania infection.
1. Introduction
The microbiota is the collective populations of bacteria, viruses,
fungi, protozoa and archaea found in our environment or associated
with various tissues and organs throughout our body. It has been esti-
mated that there are from 3 to 10 times more bacterial cells in the body
than human cells, and it is evident that the microorganisms associated
with our body are important players in our biology. Bacteria are found
in or on many parts of the body, including the intestinal tract, skin,
mouth, and the reproductive tract. While the exact numbers may vary
depending on size and gender of the person, early studies suggested that
the intestinal tract harboured the most bacteria with about 10
followed by the skin with about 10
cells, while the rest of the body
sites harbor around 10
bacteria combined [1,2]. Many studies have
focused on the bacteria in the intestinal tract, but recently studying the
commensal bacteria on the skin has become a broader area of interest.
Prior to the age of genomics, culture based methods were used to study
the bacteria in the environment [3]. However, it became apparent that
simply culturing samples was not capturing all the bacteria present
[4,5]. The discovery that bacterial phylogeny could be determined
based on the well-conserved 16S ribosomal RNA (rRNA) gene [6] set
the stage for the present-day microbiota studies. Presently, bacterial
communities are identied using high-throughput sequencing. Studies
have shown that there is lot of diversity in healthy microbiota and
perturbations in this microbiota called as dysbiosisare usually
associated with inammation and various diseases such as cancer, in-
fectious diseases, and metabolic disorders [7,8]. While many of these
studies show only correlations between dysbiosis and disease, more
recent research has focused on determining whether dysbiosis is a cause
or consequence of disease. Various studies focusing on intestinal tract of
humans during diseased state and normal state have shown that the
dysbiosis in intestinal bacterial population leads to drive disease in
arthritis, obesity, cancer, and colitis [912]. This outcome is mediated
through immunomodulatory response. However, some studies have
shown a completely opposite data indicating that this dysbiosis can
evoke an immune regulatory phenotype for protection against disease
[13]. Few studies have been conducted to analyse the co-relation of skin
microbiota and diseases. Based on above studies, it is clear that Site
specic microbiota has a dened role in diseases. This led us to the
idea of understanding this in Leishmania disease model where based on
species disease has a dierent pathological site varying from visceral
organs to skin viz, Leishmania major causing Cutaneous Leishmaniasis
and Leishmania donovani causing Visceral Leishmaniasis. The present
review summarises the site specic microbiota, their role in disease
pathology and immunomodulation taking Leishmaniasis as disease
model systems.
1.1. Skin
Skin is usually termed as First Line of Defense. It serves as a
Received 26 July 2019; Received in revised form 17 September 2019; Accepted 2 October 2019
Corresponding author.
E-mail address: (S. Singh).
Immunology Letters 216 (2019) 79–88
Available online 31 October 2019
0165-2478/ © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
Table 1
Distribution of species along diversied microbiome.
S.No. Bacterial Species Sample Site Laboratory model Human Sandy
1. Streptococcus
Enterococcus spp.
Staphylococcus aureus
LCL lesions Skin [31]
2. Enterobacter sp
Proteus sp
Pseudomonas aeruginosa
single lesions Skin ([32])
3. staphylococcus aureus
coagulase negative Staphylococcus
E. coli
Proteus sp. Klebsiella sp.
CL lesions Skin [33]
4. Staphylococcus
Providencia Peptostreptococcus
chronic wounds Skin [34]
5. Staphylococcus Streptococcus Corynebacterium,
LCL Lesions Skin [3]
6. Prevotella (2 + 7+9)
Ruminococcaceae UCG-002
Clostridialesvadin BB60 group
Ruminococcaceae UCG-014
VL patient and endemic contact faeces Gut [78]
7. Anoxybacillus
Bacillus clausii
Bacillus mycoides
Micrococcus tetragenes
Staphylococcus cohnii
Staphylococcus nepalensis
dissected midguts Gut gut (P. argentipes)
(continued on next page)
P. Misra and S. Singh Immunology Letters 216 (2019) 79–88
primary host for various microbes such as bacteria, fungi and viruses. It
has been scientically proven that these microbes termed as skin mi-
crobiome play an important role in healing of wounds, protecting from
various infectious agents, initial inammatory immune response and
allergic reactions [14,15]. Various diseases have been co-related to
changes in skin microbiota [8,16]. How the changes in these microbiota
aect the disease as well as role of these microbes in modulating the
dermal cell responses is also not explored a lot. Herein, we are
Table 1 (continued)
S.No. Bacterial Species Sample Site Laboratory model Human Sandy
8. Alcaligenes faecalis
Bacillus rmus
Bacillus exus
Bacillus mojavensis
Bacillus pumilus
Bacillus vallismortis
Enterococcus gallinarum
Bacillus altitudinis
Bacillus amyloliquefaciens
Bacillus brevis
Bacillus cereus
Bacillus circulans
Bacillus endophyticus
Escherichia blattae
Bacillus licheniformis
Oceanobacillus species
Proteus mirabilis
Proteus vulgaris
Providencia rettgeri
Pseudomonas aeuroginosa
Pseudomonas geniculate
dissected midguts Gut Sandy(P papatasi)
9. Acinetobacter junii
Cedecea Sp.
Citrobacter Sp.
Diploricketseilla Sp.
Erwinina sp.
Escherichia sp.
Pantoea sp.
Pluralibacter sp.
Pseudomonas marginalis
Pseudomonas sp.
Pseudomonas trivialis
Rickettsia sp.
Rickettsiella sp.
Spiroplasma sp.
Wolbachia sp.
dissected midguts Gut (P Chinese
10. Dissected midguts Gut P dubosqi
11. Bacillus casamanesis
Bacillus galactosidilyticus
Bacillus olironius
Bordetella avium
Brevundimonas terrae
Burkholderia fungorum
Ehrlichia sp
Kocuria polaris
Lysinibacillus sp
Microbacterium sp
Micrococcus sp
Nocardia ignorata
Ochrobactrum intermedium
Rhizobium pusense
Saccharomonaspora sp
Sporosarcina koreensis
Wolbachia inokumae
Dissected midguts Gut P pernicious
12. Asaia sp. Dissected midguts Gut P. sergenti
P. Misra and S. Singh Immunology Letters 216 (2019) 79–88
discussing site specic microbiome in Leishmaniasis.
1.1.1. Skin microbiome and cutaneous leishmaniasis
When it comes to skin, the form of Leishmaniasis which is being
manifested at this local site is cutaneous leishmaniasis (CL). The disease
is basically divided into three phenotypes based on its clinical mani-
festation from self-healing to chronic/metastatic lesions [17]:(I) Loca-
lized CL(LCL), characterized by painless ulcerative lesion [3] which
may vary from a single lesion to many (II) Muco-cutaneous Leishma-
niasis characterized by destructive mucosal lesions (III) diuse CL
(DCL), presenting multiple non-ulcerative nodules. The disease spreads
by the bite of infected sand-y[1820]. The variation of disease from
being localized to chronic or metastatic has been explored less, how-
ever, few studies says that it is not due to rigorous replication of the
parasite but the inated immune response leading to excessive in-
ammation [2126]. LCL which is said to be localised disease and self-
healing too recovers very slowly in absence of any treatment. The
available drugs have shown good response which includes pentavalent
antimonials, amphotericin B and Miltefosine [27]. The long time re-
quired for self-healing along with environmental exposure, poor hy-
gienic conditions give the way to growth of microbial population at the
site of infection. Many secondary bacterial infections have been iden-
tied in the patients and needs antibiotic treatment [28,29]. There have
been studies which have shown that disease manifestation in germ-free
mice model is dierent from that of conventional mice but how the skin
microbiota is involved in this is still unclear [15,30].
1.1.2. Characterization of skin microbiota of cutaneous leishmaniasis
Cutaneous Leishmaniasis (CL) associated microbiome studies have
been conned to very little number in LCL lesions in humans, however
culture based studies have been performed. It has been shown that
Staphylococcus spp,Streptococcus spp, Enterococcus spp, Pseudomonas spp,
and other opportunistic bacteria are present in LCL lesions
[3133].However, there has been a debate on evaluating the bacterial
composition by culture-based technique as it may compromise with the
number of species identied. This may be due to low abundance of few
species along with the dierent culture conditions required for growth
or may be the species are unculturable [34,35]. The use of massive
molecular methods allows deep insights into the microbiome-compo-
sition in general and also in the LCL microbiome because it is more
sensitive than culture, as described for other chronic wounds [34].Re-
cently comparative microbiome studies have been reported using high
throughput amplicon sequencing approach. Herein, theyhavecompared
the skin microbiota between that of laboratorial and LCL lesions with
the contralateral healthy skin(HS) microbiome from the same in-
dividuals. Restricted biological diversity was observed in LCL lesions
when compared with HS. This observation with dierence in bacterial
colonisation might be due to the fact that LCL lesion are open with a
compromised epidermis along with inammatory responses induced by
Leishmania infection leading to disturbed microbial composition [35].
The results of this study also showed that LCL lesions gets disturbed due
to contamination by commensal bacteria which were adapted to the
Leishmania induced inammation and over-growed less adapted bac-
teria [35]. They observed Actinobacteria, Firmicutes, Bacteroidetes, and
Proteobacteria [36]in HS samples which were comparable to normal
skin microbiome along with Cyanobacteria and Fusobacteria. LCL mi-
crobiome showed similar prole to non-healing foot ulcers with similar
percentage as well viz.,Firmicutes(67 %), Actinobacteria(14 %),Proteo-
bacteria(9.8 %),Bacteroidetes(7.3 %), and Fusobacteria (1.4 %) at phylum
level [37].Aerobic bacteria including Lactobacillus and Pseudomonas
which play protective role in skin by production of lactic acid and other
anti-microbial compounds were found to be decreased in LCL [38]. Few
new bacterial species such as (Fusobacterium, Bacteroides, and Peptoni-
philus), microaerophiles, and facultative anaerobic (Streptococcus, Sta-
phylococcus, Morganella, Campylobacter, and Arcanobacterium) bacteria
were most frequently detected in LCL. Table 1 enlists all those bacterial
species involved in diversied microbiome.
1.2. Immune system and skin microbiome in context of Cutaneous
Various studies have established the co-relation between changing
skin microbiota and disorders associated with skin for e.g. atopic der-
matitis, psoriasis, and chronic diabetic wounds [8,16,36,39]. However,
the question that what causes the changes is still unanswered? Reports
suggest that alike to gut microbiota, the microbial ora in skin can
modulate the immune responses in skin which promote the defense
mechanism against the pathogen and alleviate the inammatory re-
sponse to maintain the homeostasis in tissue. It has been observed that
mice, in which there is no adaptive immunity, are unable to have a
control over their skin microbiota and this allows the invasion of pa-
thogens [40]. For e.g., in case of germ free mice when Staphylococcus
epidermidisis introduced, the mice restores the IL-17A production,
thereby indicating as part of skin microbiota, S. epidermidis induce Th17
cells along with other T cells that express IL-17A. It has been further
observed that Th17 cells present in skin are being modulated by site
specic skin microbiota, without having any modulation due to gut
microbiota, this suggests that immune responses are controlled in a
compartmentalized manner [41,15].
During inammation, cytokines, chemokines, and antimicrobial
peptides are often produced, potentially explaining why there are
changes in the microbiota. Table 2 talks about changes in eector im-
mune responses by microbiome.
Bacteria present in the microbiota such as Salmonella typhimurium
and E. coli can use these products of immune response by changing their
metabolic processes. This way of adaption to changing conditions post-
infection helps the microbiota to survive in inammatory conditions
[4244]. Above-said has been well established in case of intestine [42],
but much is not explored in case of skin. However, it is clear that skin
microbiota can modulate the cutaneous immune response.
It has been reported that microbes present in skin microbiota such
as Staphylococcus can evoke Th1/Th17 immune response in the skin. T-
cell response in skin occurs by synchronized activation of skin-resident
dendritic cells. This indicates that site specic cells present in particular
tissue are tuned to respond in case of changes in microbial population.
The skin immune system response can help in protection from the pa-
thogen in some cases, conversely, it can drive the inammatory re-
sponse in other pathogens such as L. major [15]. The microbial popu-
lation plays a very important role in immune response, as these may
help in developing regulatory responses at an early age which can
protect from inammation at a later stage [45].
Various studies have suggested that the microbial population in the
skin can inuence the skin immunity, but it has been less explored that
the imbalance or Dysbiosisin these bacteria can aect disease pro-
gression, if any. In case of atopic dermatitis, it has been shown that the
dysbiosis can promote the disease progression [46]. As discussed above,
the immune system/microbiota interaction can help in disease pro-
gression or control, depending on the circumstances [13,15,46]. Post-
infection changes in skin microbiota were observed in both humans and
mice when infected with L. major. In mice, Staphylococcus spp. was
found dominantly in case of moderate lesions and higher percentage of
Streptococcus spp. in severe lesions. Gimblet et al., have further shown
that in humans dominance of both these species was found. One in-
teresting observation was that in skin during infection, innate immune
response, molecules such as antimicrobial peptides (AMPs) can target
some bacterial species and these may be involved for imbalance in skin
microbiota [38,4749]. This area is of interest and needs to be explored
more in case of Leishmania infection.
It was observed that the expression of AMP was changed post-in-
fection and mice which were decient in cathelicidin-type anti-
microbial peptide (CAMP) were more susceptible to infection. There is a
great possibility that these AMPs might cause changes in skin
P. Misra and S. Singh Immunology Letters 216 (2019) 79–88
Table 2
Changes in eector immune responses by microbiome.
Sno. Bacterial species/ severity of infection Immunological response Disease model Microbiome
1. Staphylococcus spp. dominant in moderate lesions and Streptococcus
spp. increasing in more severe lesions in group A
more neutrophils and pro-IL-1βproduction in the skin in group A A). L. major infected mice
B). Control mice
(Gimblet et al.)
2. Relative to SPF mice, GF mice infected intradermally with L. major
manifested smaller lesions with reduced edema and necrosis
GF+ S. epidermis
Impaired immune response in GF mice, with reduced- Leishmania-specic IFN-γ&
TNF-αby cutaneous T cells
Mono-associated GF mice with S. epidermidis rescued protective immunity in
these animals
A) Germ free mice
B) Special pathogen free mice
(Naik et al.)
3. Germ-free mice failed to heal lesions and presented a higher
number of parasites at the site of infection than their conventional
Germ-free mice produced elevated levels of IFN-γand lower levels of IL-4 but no
controlled infection
Isolated macrophages from Germ-free mice, exposed to IFN-γand infected with
amastigotes in vitro were not as ecient at killing parasites as macrophages from
conventional animals
** Indicates importance of microbiota in macrophage activation
A) Swiss/NIH germ-free mice
B) Conventional (microbiota-bearing) mice
**Both infected with Leishmania major
4. Clostridia (phylum Firmicutes) and Gammaproteobacteria (phylum
Actinobacteria and Bacteroidia classes
IL-1βpositively co-related with most of the microbial classes in the self-healing
mice but only with Bacilli and Gammaproteobacteria in non-healing mice
IL-12 and Il-10 have the most immune-microbial correlations out of all the
cytokines in the non-healing mice
Strong positively correlation with IL-10 levels which suggested that these
bacteria may be responsible for exerting an IL-10 dependent anti-inammatory
eects on the host
C57BL/6 (resistant)
BALB/c (susceptible) mice
**both infected with L. major
Gut of mice(faeces
5. Gut microbes from the sand y are egested into host skin alongside
Leishmania parasites
Egested microbes triggered inammasome and produced IL-1β, which sustains
neutrophil inltration.
Reducing midgut microbiota by pretreatment of Leishmania-infected sand ies
L. donovani-infected sand ies harboring transmissible
infections reproducibly transmit about 10
Gut (Sand-y)
(continued on next page)
Table 2 (continued)
Sno. Bacterial species/ severity of infection Immunological response Disease model Microbiome
with antibiotics, or neutralizing the eect of IL-1β, in bitten mice abrogates
neutrophil recruitment.
parasites to mice ears
(Dey et al.)
P. Misra and S. Singh Immunology Letters 216 (2019) 79–88
microbiota but the mechanism still needs to be elucidated. It is well
established that virulent factors can help in resistance for AMPs in case
of bacteria and both the species Staphylococcus spp. and Streptococcus
spp. found in abundance in skin site express the genes required for
protecting them from AMP [5052]. This might have helped in survival
of both bacterial species during Leishmania infection. It was also
proven using mouse with dysbiotic skin microbiota, that naturally ac-
quired dysbiosis can cause change in inammatory responses and dis-
ease progression in Leishmania. There are evidences suggesting that
environmental conditions aect skin microbiota [53]. Data suggested
that Leishmania infection disturbs the rst defense system i.e., skin and
also causes dysbiosis in skin microbiota. This concluded a hypothesis
which was of great attention that this dysbiosis caused due to Leish-
mania infection leads to recruitment of neutrophils and IL-1βrecruiting
cells to the skin, and causes increased lesion severity [23,25,54,55].
1.3. Gut microbiome
1.3.1. Gut microbiota and leishmaniasis
Various studies have been carried out to understand the eect of
bacteria in intestinal tract causing dysbiosis in disease model systems
such as arthritis, obesity and cancer [9,10,12].It has been found that the
eect of microbiota on the disease outcome is through modulation of
immune response. There are reports suggesting both way through, i.e.,
the immune system may go along with the intestinal microbiota to ei-
ther enhance the disease, protect against the disease or help in pro-
tecting host from inammatory responses [13]. It has been reported
that gut microbiome have the ability to modify the immune responses
by various mechanisms including activating macrophages and eecting
the dierentiation of T-cells. For animal model studies, usually germ
free mice are used for study of microbiota because of the fact that in
mice susceptible to systemic infection, there is a strong possibility that
the parasite can modulate the host-microbiota [56]. The germ free mice
would either be more susceptible or resistant to the disease [57]. Very
few studies have been conducted on this aspect in Leishmaniasis. Oli-
veira et al. [30], has conducted the study using Swiss/NIH germ-free
mice and microbiota bearing normal mice to understand the eect of
host microbiota on T cell dierentiation post infection with Leishmania
major. They have shown that there was no lesion healing in germ free
mice along with a higher parasitic load at the site of infection than their
conventional counterparts. The initial levels of cytokine IL-2, IL-12 and
IFN-γwas nearly equal to their control group i.e, conventional mice.
During the course of infection germ free mice produced high levels of
IFN-γand lower levels of IL-4. The data suggested that Th-1 response
was induced in the germ free mice as well. However, it was found that
macrophages isolated from germ free mice and stimulated with IFN-γ,
were not able to kill the Leishmania parasite once infected with them
concluding the fact that microbiota eected the activation of macro-
phages without having any eect on Th1 response. This indicates the
correlation between gut microbiota and modulation of the immune
response during Leishmania infection, which may nally change the
disease outcome.
Recently, a study used meta-taxonomic analysis to analyse the
faecal samples of individuals from endemic areas for visceral leishma-
niasis (VL) in India to determine the composition of gut prokaryotic and
eukaryotic microora and nd a co-correlation with diseased and non-
diseased state by comparing the dierence between VL cases and non-
VL endemic controls. High abundance of Escherichia-Shigella (9.1 %
aggregate abundance) was obtained from bacterial microbiota in a
subset of individuals that could dene a dierent enterotype or sub-
enterotype in the population (North-East India endemic) for VL com-
pared to other Indian studies. It was inferred that high Escherichia-
Shigella was associated with an overall dysbiosis of the gut bacterial
ora [78].
A study was conducted to verify the fact that does the diverse
bacterial population in the gut eects colonization of pathogens
negatively. Phlebotomus duboscqi was treated with antibiotic with an
aim of improving their vector competency, however it resulted into ies
which were refractory to the development of transmissible infection
and it happened because in the treated group parasites were not able to
dierentiate into the infective, metacyclic stage. Once these ies were
fed with dierent symbiont bacteria, the defect in parasite development
was overcome. It was also observed that when the antibiotic treated
ies were given low sucrose concentration meals, the inhibitory eect
of antibiotic treatment was moderate. These observations suggested
that competing with microbiota for sucrose utilization, produced ap-
propriate nutrient stress along with osmotic conditions for stage dif-
ferentiation and survival of infectious metacyclic promastigotes in vivo.
Most of the studies in gut microbiota and Leishmaniasis have been
focused on gut microbiota of the disease vector that are sandies. In this
section, we are focusing on various aspects of gut microbiota of sand-
1.4. Gut microbiome of Sand-y and Leishmania infection
It is a well-known fact that during Leishmania infection, parasite
resides in two forms in two hosts, one is the promastigote form which
resides in the gut lumen of the sand-y and other is the amastigote form
within macrophages of infected human host [58]. There are only few
species of sand-y which combined with Leishmania species transmit
metacyclic parasites to human host which include L. donovaniPhle-
botomusargentipes, L. majorP. papatasi,L. tropicaP. sergenti [59].
Leishmania parasites which have been taken along with the blood meal
escape through peritrophic matrix and get attached to mid gut epithe-
lium [60]. It has been well proven that gut of insects are rich in com-
mensal bacteria [61]and in case of mosquitoes and tsetse ies these
microbiota aects the ability of insects as vector for the disease [62,63].
2. Characterization of gut microbiome of the sand-y
The symbiotic microorganism present in vector causing disease af-
fects various aspects of vector which may include reproduction, nutri-
tion and homeostasis of immune system. This microbiota present in
vector along with aecting the vector can also hamper its ability for
pathogen transmission by inducing various factors such as innate de-
fence molecules, enzymes and toxins [63]. Sand-y phlebotomine
which is a vector for transmission of Leishmaniasis might acquire the
microbiota from soil, plants and since the life cycle of Leishmania
parasite in the invertebrate host occurs in digestive tract, there is a
strong possibility of interaction of various stages of Leishmania parasite
with the gut microbiota.
A study based on this hypothesis for Leishmania has addressed the
question that Does Sand-y helps in developing infectious Leishmania
parasites for transmission to host?They used comprehensive 16SrDNA
gene high-throughput sequencing of DNA obtained from the dissected
midguts of infected or uninfected Lutzomyia longipalpis, which transmits
Leishmania infantum. Nearly 2091 Lu. Longipalpis were used to isolate
midgut and varied array of bacterial species were identied. These
species varied based on the source of the insects meal and infection
status. It was found that there was a drastic and regular loss in bacterial
community post L. infantum infection as the infection progresses [64]. It
was observed that bacteria of phylum diered signicantly in micro-
biome of infected sand-ies from that of uninfected controls fed on
either blood or sucrose along with bacteria from the family Phyllo-
bacteraceae and the genus Trabulsiella in both the groups. They also
observed prominent presence of Enterobacteriacae under both sucrose-
fed and blood-fed conditions which were surpassed by Acetobacteraceae,
12 days post infection. Other studies which are based on dierent
identication methods including denaturing gradient gel electrophor-
esis (DGGE), bacterial culture have also identied various bacterial
species [65]. The denaturing gel electrophoresis performed with DNA of
Lu. longipalpis or Lu. Cruzit aken from regions of rural orsylvatic Brazil
P. Misra and S. Singh Immunology Letters 216 (2019) 79–88
or Colombia identied Proteobacteria Erwinia and Ralstonia spp.[66].
Sequencing of midgut metagenome of Lu. intermedia which is the vector
for L. brazilienses was performed by Monteiro et al. They identied
Enterobacteriacae in both the uninfected and gravid groups but re-
presented only 4.2 %of the bacterial families of blood-fed groups.
Rickettsiaceae was found in most abundance in Lu. intermedia blood-fed
y pool of which genus Wolbachia comprised nearly 46.7 % of the
sequences. Both the Lu. intermedia and Lu. Longipalpis showed the pre-
sence of Proteobacteria and Brucellaceae along with Pseudomonas
(phylum Proteobacteria) [67]. Other species of sand-y vector were
also characterized. The microbiota composition in the midgut of P.
perniciosus revealed that lab-reared P. perniciosushad less rich bacterial
microbiome in midgut than in eld-collected sand ies which might be
due to dierence in food intake. P. perniciosus midgut, contained bac-
teria belonging to Burkholderia genus and Stenotrophomonas maltophilia.
They also identied few bacterial species which are not characterized in
sand-y midgut but are present in human or other mammalian midgut
like Veillonella addition to Sporosarcinakoreensis, Rhizobium pusense
and Nocardia. The dierence in midgut microbiota of Lutzomyia sp. and
P. perniciosus was attributed to enormous factors which included long
divergence of evolution between the two subgenera [68].
3. Sand-y microbiota and Leishmania
We have discussed that Leishmania infection in midgut of sand-yis
accompanied by gut microbiota which has predominance of bacterial
species. It has been suggested that microbiota has an important eect
on the physiology of any disease transmitting vector and also inuence
the innate immune system [63]. There are various reports suggesting
that microbiota can activate the innate immune pathway, thereby af-
fecting the parasitic infection and induce some eector molecules
which can control the infection. It has been shown that the suppression
of ROS in midgut of L. longipalpis facilitates the leishmania infection
proving the importance of microbiota [69]. In line with observation in
other vector parasite disease models, the competency of sand-y is in-
uenced by microbiota in Leishmania infection as well [70,71]. It was
found in experiments conducted with colony-raised L. longipalpis, that if
the insects are feeded with bacteria Asaia sp., Ochrobactrumintermedium
and a yeast-like fungus Pseudozyma sp. which were isolated from
midgut of wild and laboratory reared female L. longipalpis before
leishmania infection, the disease does not get established [66]. It was
veried in the same study that once infected with L. mexicana, L.
longipalpis was resistant to Serratia infection. Many studies have further
shown that the development of promastigote stage of Leishmania
parasite in the vector sand-y is dependent on the microbiota of the
vectors midgut. Study carried out by Kelly et al. [64]rst characterized
the phylogenies of bacteria present within the midgut of three cate-
gories of sand-yL. longipalpis i.e, sugar-fed, blood-fed and L.infantum-
infected. This observation was also accompanied by observing the eect
of treatment by antibiotic against Leishmania infection. The results
showed that once infected with Leishmania parasite, the diversity of
bacterial population in midgut of L. longipalpis was lost gradually;
moreover, the treatment with antibiotic aected the replication of L.
infantum and its metacyclic form. More or less similar observation was
found Louradour et al. [72], where they have shown that development
of L. major in P. duboscqiis is hampered by antibiotic treatment. The fact
that microbiota of sand-y has a role in Leishmania development and
replication in midgut was further proved by using engineered anti-
biotic-resistant bacteria isolated from natural P. duboscqi and in this
condition antibiotic treatment did not aect the Leishmania infection in
the sand-y midgut [66]. The importance of exploring the area of sand-
y microbiota in Leishmaniasis was strengthened again by the work
carried out in Leishmania donovani infected mammals by Dey et.,al.
which would be subsequently discussed in the next section [73].
3.1. Gut microbiota and immune system interplay
An extensive study carried out by Lamour SD et al. [74]on mouse
microbiota has co-related the microbiota with the cytokines produced.
They have investigated the eect of L. major infection on microbiota of
C57BL/6 (resistant) and BALB/c (susceptible) mice. They found that
faeces of both the mice models contained bacteria mainly from two
classes i.e. Clostridia (phylum Firmicutes) and Gammaproteobacteria
(phylum Proteobacteria). It was found that Clostridium increased sig-
nicantly post infection in BALB/c mice than in C57BL/6 mice which
was initially similar. Initially post 2 weeks of infection in both animal
models, Gammaproteobacteria decreased but at the time of termination
of experiment it was found that this bacterium was signicantly higher
in the resistant mouse strain. This higher presence of Gammaproteo-
bacteria class was correlated with resistance in C57BL/6 to infection
with L. major based on the observation that although levels of Gam-
maproteobacteria decreased in both the mice models at the end of the
study but the levels of this bacteria in C57BL/6 mice remained sig-
nicantly higher than in BALB/c mice.
When cytokine prole was co-related with the microbial population
it was found that IL-1βhas shown positive co-relation with most of the
microbial classes in the self-healing mice but co-related only with Bacilli
and Gammaproteobacteria in non-healing mice. Similarly, it was found
that IL-12 and Il-10 have the most immune-microbial correlations out of
all the cytokines in the non-healing mice. Two bacterial classes namely,
Actinobacteria and Bacteroidia classes showed a very strong positively
correlation with IL-10 levels which suggested that these bacteria may
be responsible for exerting an IL-10 dependent anti-inammatory ef-
fects on the host. This prole shows an extensive and strong co-relation
between immune response and microbiota in self-healing mice which
was dierent from non-healing mice which might explain the dierence
in two animal models for Leishmania infectivity. Since above section
has shown the importance of sand-y microbiota in Leishmania infec-
tion, we focused on this and found that recently a group has given a
fundamental role of sand-y microbiota in Leishmania infection in
mammalian host [73]. They have shown that when L. longipalpis sucks
the blood meal for infection than along with the parasite, vector mi-
crobiota is also egested with the parasite inoculum. The microbe po-
pulation of sand-y leads to activation of the neutrophil inammasome
of mouse and further results in a rapid production of interleukin-1β(IL-
1β), which sustains neutrophil inltration. Due to these neutrophils
Leishmania donovani parasites are shielded and this helps in promoting
infection of macrophages post transmission. Impairing the sand-y
microbiota by antibiotic treatment aects the infection of Leishmania
donovani. This data gave a new insight that sand-y midgut microbiota
can not only aect the Leishmania within it, but these micro-organisms
when present modulate the host immune response in favor of the
parasite transmission and survival.
Another work based on the fact that LACK-specic Tcells accumu-
late IL-4 mRNA very rapidly in mice infected with L.major, character-
ized the phenotype of cells before infection. They demonstrated a very
interesting fact that the lymphoid organs of naive BALB/c mice were
found to have microbial Ag-specic T cells which had the ability to
cross-react with LACK and that express a memory/eector phenotype.
This could have been the reason for secretion of IL-4 shortly after in-
fection by LACK-specicT cells.The group incubated ve dierent
LACK-specic Tcell hybridomas with crude extracts from various
aerobic and anaerobic bacteria extracts and it was found that
Escherichia coli and Enterococcus faecalis, but not Proteus mirabilis and
Clostridium perfringens extracts induced secretion IL-2 by T-cell hy-
bridomas. Overall, data suggested that the IL-4 burst induced by
parasite on priming of LACK-specicT cells is due to microbial Ags,
Theseare among very few studies which has shown the eect on sys-
temic Ag-specic immune responses mediated by intestinal ora and
suggests that the immune response generated against the cutaneous
parasite may be due to cross-priming of T-cells by microbial Ags from
P. Misra and S. Singh Immunology Letters 216 (2019) 79–88
the indigenous intestinal ora [75].
4. Metabolites and microbiota interplay in Leishmaniasis
Metabolic proling reveals an insight into the altered host micro-
biome across diverse sets of parasite-rodent models evident through a
set of microbiota-associated metabolites in urine and plasma. To eval-
uate the importance of the host microbiome on the immune response
specically dierentiation of T cell subsets during the course of infec-
tion, assessment of various parameters such as lesion development,
parasite loads, and cytokine production was carried out in Swiss/NIH
germ-free mice and conventional (which has microbiota) mice.Despite
showing strong Th1 immune response in L.major infection models, re-
sults are indicative that germ-free mice failed to heal lesions in com-
parison to their conventional counterparts [30]. Having cited that, the
study demonstrates intermittent role of microbiota in mounting a suc-
cessful host response to the parasite.
In general, when any parasite gets introduced in the mammalian
system, it might disrupt the microbiota present and this may lead to
changes in metabolism. In various studies, when the parasite and rodent
model was characterized, a strong co-relation was observed between
the metabolites, altered host microbiome and infection. These data are
based on the metabolite proling in conjunction to associated micro-
biota either in urine or plasma [76]. Metabonomics approach was used
in case of Schistosoma mansoni infection in mice for characterizing the
intergenome interaction between the gut microora and infection. They
found many microbial population related metabolites also such as tri-
methylamine, phenylacetyl glycine, acetate, p-cresol glucuronide, bu-
tyrate suggesting that post infection there is disturbance in gut micro-
biota. One more study conducted by Dumas et al. [77] has shown a
complex interaction between the gut microbiota and host co-metabo-
lites in impaired glucose homeostasis induced by diet, and non-alco-
holic fatty liver disease (NAFLD), in the strain of susceptible mouse. The
samples of plasma and urine were collected for the study using
Their data indicated that host metabolism is altered due to the altera-
tion in metabolism of gut microbiome. NAFLD is associated with the
disturbances in choline mechanism, wherein, there are low levels of
plasma phosphatidylcholine in circulation and urine consists of high
levels of methylamines which are co-processed by gut microbiota and
mammalian enzyme systems. This reduction in choline which was
mimicked by choline-decient diet caused NAFLD. Having said that, it
strongly indicates the prominent role of gut microbiome in insulin re-
sistance [77]. In case of Leishmania major, it has been observed that
inspite of generating a strong Th1 type protective response, the germ
free mice were not able to heal the lesions when compared to their
normal counterparts [30].The data suggests strong role of microbiota in
elucidating a strong host response to the infection. Another study was
performed to evaluate the response to infection in a self-healing C57BL/
6 and a non-healing BALB/c mice model for cutaneous leishmaniasis.
They combined three important aspects to evaluate the outcome viz.,
immune response, metabolic outcomes and gut microbiota response in
the host. Urine, plasma and faeces were included for metabolic pro-
ling, peripheral cytokines and faecal bacterial constituents were ana-
lysed [74]. The study identied a strong co-relation between im-
munological response, metabolic prole and microbiota in L. major.
Direct statistical interaction was observed after correlation network
analyses using metabolome of host, cytokines and the microbial com-
position of faeces. It was found that self-healing strain has more number
of co-relations among the above-said parameters whereas non-healing
mice did not have.
4.1. Skin microbiome and gut microbiome interaction
Very few studies have been done to understand the role of the in-
digenous microbiota during Leishmania infection. It would be inter-
esting to study the dynamic interaction between the host, disease site
and the microbiota. We envisioned through systems biology approach
to churn some bacterial species in skin microbiota and Phlebotmus gut
microbiota during Leishmania infection, but surprisingly we did not nd
any common species inspite of the fact that both the sites are involved
in disease manifestation. Supplementary Table mentions the con-
nectome of the microbiome constructed with their corresponding
Pubmed ID. This indicates that identifying a single bacterium that can
be applied to control strategies targeted to a majority of sand-y vec-
tors, skin manifestation of disease will be quite challenging (Fig. 1).
Community level modularity in dierent microbiomes may be asso-
ciated with decreased level of variability in the gut environment or with
the lack of temporal regularities. Integrated computational-microbiome
model may ultimately help devise a predictive framework for targeted
community manipulation in disease model systems and even for
Fig. 1. Connectome of the Microbiome.
P. Misra and S. Singh Immunology Letters 216 (2019) 79–88
informing clinical interventions. To determine which aspects of gut
microbiome may contribute to disease and decipher the mechanism
linking to host pathophysiology and immunity may shed some im-
portant scientic questions to ponder upon in future.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inu-
ence the work reported in this paper.
We are thankful for the intramural support, provided by Department
of Biotechnology, Ministry of Science and Technology, Government of
India. PM acknowledges her nancial support from Council of Scientic
and Industrial Research too.We are also thankful to the Director,
National Centre for Cell Science (NCCS) for supporting the
Bioinformatics and High Performance Computing Facility (BHPCF) at
NCCS, Pune, India
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:
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... It has also been shown that germ free mice have an impaired immune response against Leishmania parasites that can be partially rescued by inoculation of the commensal skin bacteria, S. epidermidis (273). Leishmania might induce dysbiosis; this rupture in skin homeostasis would lead to the recruitment of neutrophils and IL-1 beta secretion increasing the severity of the disease (275). Surprisingly, up to now no such studies have been performed on ticks and tick-borne pathogens to analyze the role of host microbiota in tick-attractiveness and pathogen transmission. ...
... What is the role of the microbiota in the case of pathogens co-inoculated with tick saliva? Some preliminary data exists for certain VBDs like malaria (320) and leishmaniasis (275), but none for TBDs. In malaria, the skin microbiome is clearly involved in attractiveness of mosquito and the intestinal microbiome of the vertebrate host seems also to influence the outcome of the disease, at least in mouse model. ...
... It has also been shown that germ free mice have an impaired immune response against Leishmania parasites that can be partially rescued by inoculation of the commensal skin bacteria, S. epidermidis (273). Leishmania might induce dysbiosis; this rupture in skin homeostasis would lead to the recruitment of neutrophils and IL-1 beta secretion increasing the severity of the disease (275). Surprisingly, up to now no such studies have been performed on ticks and tick-borne pathogens to analyze the role of host microbiota in tick-attractiveness and pathogen transmission. ...
... What is the role of the microbiota in the case of pathogens co-inoculated with tick saliva? Some preliminary data exists for certain VBDs like malaria (320) and leishmaniasis (275), but none for TBDs. In malaria, the skin microbiome is clearly involved in attractiveness of mosquito and the intestinal microbiome of the vertebrate host seems also to influence the outcome of the disease, at least in mouse model. ...
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Ticks and tick transmitted infectious agents are increasing global public health threats due to increasing abundance, expanding geographic ranges of vectors and pathogens, and emerging tick-borne infectious agents. Greater understanding of tick, host, and pathogen interactions will contribute to development of novel tick control and disease prevention strategies. Tick-borne pathogens adapt in multiple ways to very different tick and vertebrate host environments and defenses. Ticks effectively pharmacomodulate by its saliva host innate and adaptive immune defenses. In this review, we examine the idea that successful synergy between tick and tick-borne pathogen results in host immune tolerance that facilitates successful tick infection and feeding, creates a favorable site for pathogen introduction, modulates cutaneous and systemic immune defenses to establish infection, and contributes to successful long-term infection. Tick, host, and pathogen elements examined here include interaction of tick innate immunity and microbiome with tick-borne pathogens; tick modulation of host cutaneous defenses prior to pathogen transmission; how tick and pathogen target vertebrate host defenses that lead to different modes of interaction and host infection status (reservoir, incompetent, resistant, clinically ill); tick saliva bioactive molecules as important factors in determining those pathogens for which the tick is a competent vector; and, the need for translational studies to advance this field of study. Gaps in our understanding of these relationships are identified, that if successfully addressed, can advance the development of strategies to successfully disrupt both tick feeding and pathogen transmission.
... Cell-mediated immune responses associated with protection in the skin of CL patients CL in NA and FG is characterized by various immunological features ( Figure 3) (44,47,58,74,(88)(89)(90). Once the Leishmania infected sandfly bites, the infection starts with an asymptomatic "silent phase" of variable duration, characterized by an inflammatory wave of poly morpho nuclear (PMN), dendritic cells (DC), and monocyte-derived macrophages which harbour a proliferation of amastigotes intracellular parasites (10,91,92). Then, a massive recruitment of CD4 + and CD8 + T lymphocytes, with enhanced pro-inflammatory responses participate in granuloma formation and parasite control. ...
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Cutaneous leishmaniasis (CL) caused by infection with the parasite Leishmania exhibits a large spectrum of clinical manifestations ranging from single healing to severe chronic lesions with the manifestation of resistance or not to treatment. Depending on the specie and multiple environmental parameters, the evolution of lesions is determined by a complex interaction between parasite factors and the early immune responses triggered, including innate and adaptive mechanisms. Moreover, lesion resolution requires parasite control as well as modulation of the pathologic local inflammation responses and the initiation of wound healing responses. Here, we have summarized recent advances in understanding the in situ immune response to cutaneous leishmaniasis: i) in North Africa caused by Leishmania (L.) major, L. tropica, and L. infantum, which caused in most cases localized autoresolutives forms, and ii) in French Guiana resulting from L. guyanensis and L. braziliensis, two of the most prevalent strains that may induce potentially mucosal forms of the disease. This review will allow a better understanding of local immune parameters, including cellular and cytokines release in the lesion, that controls infection and/or protect against the pathogenesis in new world compared to old world CL.
... Immunity against Leishmania is complex and depends on many factors, such as genetic diversity, parasite species and isolates (27)(28)(29). Leishmania spp. are inoculated into the skin as metacyclic promastigotes (30) and once the parasites are in close contact with the body, immunity is triggered (Figure 1). ...
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Leishmaniasis are Neglected Tropical Diseases affecting millions of people every year in at least 98 countries and is one of the major unsolved world health issues. Leishmania is a parasitic protozoa which are transmitted by infected sandflies and in the host they mainly infect macrophages. Immunity elicited against those parasites is complex and immune checkpoints play a key role regulating its function. T cell receptors and their respective ligands, such as PD-1, CTLA-4, CD200, CD40, OX40, HVEM, LIGHT, 2B4 and TIM-3 have been characterized for their role in regulating adaptive immunity against different pathogens. However, the exact role those receptors perform during Leishmania infections remains to be better determined. This article addresses the key role immune checkpoints play during Leishmania infections, the limiting factors and translational implications.
Cutaneous Leishmaniasis (CL) affects millions of people globally and has a significant impact on morbidity and mortality. Innate immune mediators are likely to influence the clinical phenotype of CL through primary responses that restrict or facilitate parasite spread. The aim of the study was to bring to attention the significance of microbiota in the development of CL and emphasized the necessity of including the role of microbiota in CL while promoting a One Health approach for managing diseases. To achieve this, we used 16S amplicon metagenome sequencing and QIIME2 pipeline to analyze the microbiome composition of CL-infected patients compared to non-infected, healthy subjects. 16S sequencing analysis showed serum microbiome was dominated by Firmicutes, Proteobacteria, Bacteroidota, and Actinobacteria. CL-infected individuals, Proteobacteria were the most prevalent (27.63 ± 9.79), with the relative abundance (10.73 ± 5.33) of Proteobacteria in control. Bacilli class was found to be the most prevalent in healthy controls (30.71 ± 8.44) while (20.57 ± 9.51) in CL-infected individuals. The class Alphaproteobacteria was found to be more in CL-infected individuals (5.47 ± 2.07) as compared to healthy controls (1.85 ± 0.39). The CL-infected individuals had a significantly lower relative abundance of the Clostridia class (p < 0.0001). An altered serum microbiome of CL infection and higher microbial abundance in the serum of healthy individuals was observed.
The ectoparasites Dactylogyrus lamellatus usually infest fish gills and cause severe damage to the gill tissues of the host. However, whether the ectoparasites influence gut microbiota is still unknown. In this study, gut microbiota combined with histopathological characteristics, biochemical parameters, and relative expression of immune-related genes were analyzed in grass carp severely infected (SI), mildly infected (MI) and uninfected (UI) with D. lamellatus. Deep amplicon sequencing of the 16S rRNA gene of the gut microbiota revealed that the diversity of the SI group was significantly reduced than UI group (p < 0.05). In parallel with the increased loads of D. lamellatus, the relative abundance of Cetobacterium increased, and that of Bacteroides decreased. Antioxidant indices of the glutathione peroxidase (GPX) in the infected groups were significantly higher than those in the UI group (p < 0.05). The expression levels of several immune-related genes encoding toll-like receptor 3 (TLR3), myeloid differentiation factor 88 (MyD88), major histocompatibility complex II (MHCII), and immunoglobulin M (IgM) were relatively increased the degree of infection in grass carp tissues. Meanwhile, the histopathological characteristics show a large number of immune cells infiltrated in the mucosal and submucosal layers in intestine. It indicates that D. lamellatus infection stimulated both the innate and adaptive immune responses. Our data demonstrated that ectoparasites D. lamellatus infection on the gills resulted in a gut microbiota shift, histopathological changes, and immune responses in the grass carp. Furthermore, ectoparasite- microbiota- host interactions indicate that fish ectoparasites probably have remote “talk” mechanisms with host intestinal microbes. This is the first study of ectoparasites on fish gills affecting the gut microbiota in grass carp from far distance away, which is beneficial for understanding the relationships between parasites, gut microbiota, and host and for the control of parasitic diseases in fish.
Leishmaniasis is a zoonotic and neglected disease, which represents an important public health problem worldwide. Different species of Leishmania are associated with different manifestations, and a practical problem that can worsen the condition of hosts infected with Leishmania is the secondary infection caused by bacteria. This review aims to examine the importance and prevalence of bacteria co-infection during leishmaniasis and the nature of this ecological relationship. In the cases discussed in this review, the facilitation phenomenon, defined as any interaction where the action of one organism has a beneficial effect on an organism of another species, was considered in the Leishmania–bacteria interaction, as well as the effects on one another and their consequences for the host.
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Visceral leishmaniasis (VL) caused by Leishmania donovani remains of public health concern in rural India. Those at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths. Intestinal helminths are potent regulators of host immune responses sometimes mediated through cross-talk with gut microbiota. We evaluate a meta-taxonomic approach to determine the composition of prokaryotic and eukaryotic gut microflora using amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) and 18S rRNA gene regions. The most abundant bacterial taxa identified in faecal samples from Bihar State India were Prevotella (37.1%), Faecalibacterium (11.3%), Escherichia-Shigella (9.1%), Alloprevotella (4.5%), Bacteroides (4.1%), Ruminococcaceae UCG-002 (1.6%), and Bifidobacterium (1.5%). Eukaryotic taxa identified (excluding plant genera) included Blastocystis (57.9%; Order: Stramenopiles), Dientamoeba (12.1%; Family: Tritrichomonadea), Pentatrichomonas (10.1%; Family: Trichomonodea), Entamoeba (3.5%; Family: Entamoebida), Ascaridida (0.8%; Family: Chromodorea; concordant with Ascaris by microscopy), Rhabditida (0.8%; Family: Chromodorea; concordant with Strongyloides), and Cyclophyllidea (0.2%; Order: Eucestoda; concordant with Hymenolepis). Overall alpha (Shannon's, Faith's and Pielou's indices) and beta (Bray-Curtis dissimilarity statistic; weighted UniFrac distances) diversity of taxa did not differ significantly by age, sex, geographic subdistrict, or VL case (N = 23) versus endemic control (EC; N = 23) status. However, taxon-specific associations occurred: (i) Ruminococcaceae UCG- 014 and Gastranaerophilales_uncultured bacterium were enriched in EC compared to VL cases; (ii) Pentatrichomonas was more abundant in VL cases than in EC, whereas the reverse occurred for Entamoeba. Across the cohort, high Escherichia-Shigella was associated with reduced bacterial diversity, while high Blastocystis was associated with high bacterial diversity and low Escherichia-Shigella. Individuals with high Blastocystis had low Bacteroidaceae and high Clostridiales vadin BB60 whereas the reverse held true for low Blastocystis. This scoping study provides useful baseline data upon which to develop a broader analysis of pathogenic enteric microflora and their influence on gut microbial health and NTDs generally.
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Leishmania donovani parasites are the cause of visceral leishmaniasis and are transmitted by bites from phlebotomine sand flies. A prominent feature of vector-transmitted Leishmania is the persistence of neutrophils at bite sites, where they protect captured parasites, leading to enhanced disease. Here, we demonstrate that gut microbes from the sand fly are egested into host skin alongside Leishmania parasites. The egested microbes trigger the inflammasome, leading to a rapid production of interleukin-1β (IL-1β), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1β in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1β are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease.
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For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.
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Importance: Leishmania infantum, a parasitic protozoan causing fatal visceral leishmaniasis, is transmitted to humans through the bite of the sand fly Lutzomyia longipalpis Development of the parasite to its virulent metacyclic state occurs in the sand fly gut. In this study, the microbiota within the Lu. longipalpis midgut was delineated by 16S ribosomal DNA (rDNA) sequencing, revealing a highly diverse community composition that lost diversity as parasites developed to their metacyclic state and increased in abundance in infected flies. Perturbing sand fly gut microbiota with an antibiotic cocktail, which alone had no effect on either the parasite or the fly, arrested both the development of virulent parasites and parasite expansion. These findings indicate the importance of bacterial commensals within the insect vector for the development of virulent pathogens, and raise the possibility that impairing the microbial composition within the vector might represent a novel approach to control of vector-borne diseases.
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Background Parasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector. Methods We performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota. ResultsBacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups. Conclusions The metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing the constituents of Lu. intermedia native microbiota contribute to the knowledge regarding the bacterial community in an important sand fly vector and allow for further studies to better understand how the microbiota interacts with vectors of human parasites and to develop tools for biological control.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
Intestinal inflammation is frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is characterized by a reduced abundance of obligate anaerobic bacteria and an expansion of facultative Proteobacteria such as commensal E. coli. The mechanisms enabling the outgrowth of Proteobacteria during inflammation are incompletely understood. Metagenomic sequencing revealed bacterial formate oxidation and aerobic respiration to be overrepresented metabolic pathways in a chemically induced murine model of colitis. Dysbiosis was accompanied by increased formate levels in the gut lumen. Formate was of microbial origin since no formate was detected in germ-free mice. Complementary studies using commensal E. coli strains as model organisms indicated that formate dehydrogenase and terminal oxidase genes provided a fitness advantage in murine models of colitis. In vivo, formate served as electron donor in conjunction with oxygen as the terminal electron acceptor. This work identifies bacterial formate oxidation and oxygen respiration as metabolic signatures for inflammation-associated dysbiosis.
Microbial burden of chronic wounds is believed to play an important role in impaired healing and development of infection-related complications. However, clinical cultures have little predictive value of wound outcomes, and culture-independent studies have been limited by cross-sectional design and small cohort size. We systematically evaluated the temporal dynamics of the microbiota colonizing diabetic foot ulcers (DFU), a common and costly complication of diabetes, and its association with healing and clinical complications. Dirichlet multinomial mixture modeling, Markov chain analysis, and mixed-effect models were used to investigate shifts in the microbiota over time and its associations with healing. Here we show to our knowledge previously unreported temporal dynamics of the chronic wound microbiome. Microbiota community instability was associated with faster healing and improved outcomes. DFU microbiota were found to exist in one of four community types that experienced frequent and non-random transitions. Transition patterns and frequencies associated with healing time. Exposure to systemic antibiotics destabilized the wound microbiota, rather than altering overall diversity or relative abundance of specific taxa. This study provides to our knowledge previously unreported evidence that the dynamic wound microbiome is indicative of clinical outcomes and may be a valuable guide for personalized management and treatment of chronic wounds.
Cutaneous leishmaniasis is a major public health problem and causes a range of diseases from self-healing infections to chronic disfiguring disease. Currently, there is no vaccine for leishmaniasis, and drug therapy is often ineffective. Since the discovery of CD4(+) T helper 1 (TH1) cells and TH2 cells 30 years ago, studies of cutaneous leishmaniasis in mice have answered basic immunological questions concerning the development and maintenance of CD4(+) T cell subsets. However, new strategies for controlling the human disease have not been forthcoming. Nevertheless, advances in our knowledge of the cells that participate in protection against Leishmania infection and the cells that mediate increased pathology have highlighted new approaches for vaccine development and immunotherapy. In this Review, we discuss the early events associated with infection, the CD4(+) T cells that mediate protective immunity and the pathological role that CD8(+) T cells can have in cutaneous leishmaniasis.