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Immunology Letters
journal homepage: www.elsevier.com/locate/immlet
Review
Site specific 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
ARTICLE INFO
Keywords:
Leishmania
Microbiome
Skin
Immune system
Gut microbiota
ABSTRACT
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 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.
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
14
cells,
followed by the skin with about 10
12
cells, while the rest of the body
sites harbor around 10
12
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 identified using high-throughput sequencing. Studies
have shown that there is lot of diversity in healthy microbiota and
perturbations in this microbiota called as “dysbiosis”are usually
associated with inflammation 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 [9–12]. 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
specific microbiota has a defined role in diseases”. This led us to the
idea of understanding this in Leishmania disease model where based on
species disease has a different 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 specific 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
https://doi.org/10.1016/j.imlet.2019.10.004
Received 26 July 2019; Received in revised form 17 September 2019; Accepted 2 October 2019
⁎
Corresponding author.
E-mail address: singhs@nccs.res.in (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.
T
Table 1
Distribution of species along diversified microbiome.
S.No. Bacterial Species Sample Site Laboratory model Human Sandfly
1. Streptococcus
Enterococcus spp.
Staphylococcus aureus
LCL lesions Skin ✓[31]
2. Enterobacter sp
Proteus sp
Pseudomonas aeruginosa
Klebsiellasp
single lesions Skin ✓([32])
3. staphylococcus aureus
coagulase negative Staphylococcus
E. coli
Proteus sp. Klebsiella sp.
CL lesions Skin ✓[33]
4. Staphylococcus
Enterococcus
Pseudomonas
Serratia
Corynebacterium
Clostridium
Bacillus
Paenibacillus
Propionibacterium
Escherichia
Streptococcus
Brevibacterium
Citrobacter
Klebsiella
Lactobacillus
Finegoldia
Sarcina
Anaerococcus
Bacteroides
Peptoniphilus
Prevotella
Enterobacter
Salmonella
Providencia Peptostreptococcus
Acinetobacter
Nocardiopsis
chronic wounds Skin ✓[34]
5. Staphylococcus Streptococcus Corynebacterium,
PeptoniphilusPeptostreptococcus,
Fusobacterium
LCL Lesions Skin ✓[3]
6. Prevotella (2 + 7+9)
Faecalbacterium
Escherichia-Shigella
Alloprevotella
Bacteroides
Ruminococcaceae UCG-002
Bifidobacterium
Roseburia
Agathobacter
Catenibacterium
Asteroleplasma
Succinivibrio
Clostridialesvadin BB60 group
Anaerovibrio
Dialister
Megamonas
Megasphaera
Mitsuokella
Lactobacillus
Ruminococcaceae UCG-014
Gastranaerophilales
VL patient and endemic contact faeces Gut ✓[78]
7. Anoxybacillus
Flavithermus
Bacillus clausii
Bacillus mycoides
Brevibacteriumcasei
Geobacillus
kaustophilus
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
80
primary host for various microbes such as bacteria, fungi and viruses. It
has been scientifically proven that these microbes termed as skin mi-
crobiome play an important role in healing of wounds, protecting from
various infectious agents, initial inflammatory 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
affect 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 Sandfly
8. Alcaligenes faecalis
Bacillus firmus
Bacillus flexus
Bacillus mojavensis
Bacillus pumilus
Bacillus vallismortis
Brevibacillusreuszeri
Brevibacteriumfrigoritalerans
Citrobactermurliniae
Enterococcus gallinarum
Bacillus altitudinis
Bacillus amyloliquefaciens
Bacillus brevis
Bacillus cereus
Bacillus circulans
Bacillus endophyticus
Escherichia blattae
Exigobacteriumindicum
Lysinibacillusboronitolerans
Bacillus licheniformis
Microbacteriumimperiale
Microbacteriumparaoxydans
Microbacteriumsediminis
Oceanobacillus species
Pantoeaananatis
Planomicrobiumglaciei
Proteus mirabilis
Proteus vulgaris
Providencia rettgeri
Pseudomonas aeuroginosa
Pseudomonas geniculate
dissected midguts Gut Sandfly(P papatasi)
9. Acinetobacter junii
Cedecea Sp.
Citrobacter Sp.
Diploricketseilla Sp.
Erwinina sp.
Escherichia sp.
Klebsiellaozaenae
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
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
Roseomonas
ludipueritiae
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
81
discussing site specific 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) diffuse CL
(DCL), presenting multiple non-ulcerative nodules. The disease spreads
by the bite of infected sand-fly[18–20]. 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 inflated immune response leading to excessive in-
flammation [21–26]. 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-
tified 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 different 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 confined 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
[31–33].However, there has been a debate on evaluating the bacterial
composition by culture-based technique as it may compromise with the
number of species identified. This may be due to low abundance of few
species along with the different 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 difference in bacterial
colonisation might be due to the fact that LCL lesion are open with a
compromised epidermis along with inflammatory 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 inflammation 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 profile 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 diversified microbiome.
1.2. Immune system and skin microbiome in context of Cutaneous
Leishmaniasis
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 flora in skin can
modulate the immune responses in skin which promote the defense
mechanism against the pathogen and alleviate the inflammatory 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
specific skin microbiota, without having any modulation due to gut
microbiota, this suggests that immune responses are controlled in a
compartmentalized manner [41,15].
During inflammation, cytokines, chemokines, and antimicrobial
peptides are often produced, potentially explaining why there are
changes in the microbiota. Table 2 talks about changes in effector 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 inflammatory conditions
[42–44]. 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 specific 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 inflammatory 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 inflammation at a later stage [45].
Various studies have suggested that the microbial population in the
skin can influence the skin immunity, but it has been less explored that
the imbalance or “Dysbiosis”in these bacteria can affect 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 [38,47–49]. 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 deficient 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
82
Table 2
Changes in effector 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.)
Skin
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-specific 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.)
Skin
3. Germ-free mice failed to heal lesions and presented a higher
number of parasites at the site of infection than their conventional
counterparts.
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 efficient 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
Skin
4. Clostridia (phylum Firmicutes) and Gammaproteobacteria (phylum
Proteobacteria)
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-inflammatory
effects on the host
C57BL/6 (resistant)
BALB/c (susceptible) mice
**both infected with L. major
Gut of mice(faeces
sample)
5. Gut microbes from the sand fly are egested into host skin alongside
Leishmania parasites
Egested microbes triggered inflammasome and produced IL-1β, which sustains
neutrophil infiltration.
Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies
L. donovani-infected sand flies harboring transmissible
infections reproducibly transmit about 10
3
–10
4
Gut (Sand-fly)
(continued on next page)
Table 2 (continued)
Sno. Bacterial species/ severity of infection Immunological response Disease model Microbiome
with antibiotics, or neutralizing the effect 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
83
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 [50–52]. 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 inflammatory responses and dis-
ease progression in Leishmania. There are evidences suggesting that
environmental conditions affect skin microbiota [53]. Data suggested
that Leishmania infection disturbs the first 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 effect 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
effect 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 inflammatory responses [13]. It has been reported
that gut microbiome have the ability to modify the immune responses
by various mechanisms including activating macrophages and effecting
the differentiation 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 effect of
host microbiota on T cell differentiation 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 effected the activation of macro-
phages without having any effect on Th1 response. This indicates the
correlation between gut microbiota and modulation of the immune
response during Leishmania infection, which may finally 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 microflora and find a co-correlation with diseased and non-
diseased state by comparing the difference 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 define a different 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
flora [78].
A study was conducted to verify the fact that does the diverse
bacterial population in the gut effects colonization of pathogens
negatively. Phlebotomus duboscqi was treated with antibiotic with an
aim of improving their vector competency, however it resulted into flies
which were refractory to the development of transmissible infection
and it happened because in the treated group parasites were not able to
differentiate into the infective, metacyclic stage. Once these flies were
fed with different symbiont bacteria, the defect in parasite development
was overcome. It was also observed that when the antibiotic treated
flies were given low sucrose concentration meals, the inhibitory effect
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 sandflies. In this
section, we are focusing on various aspects of gut microbiota of sand-
fly.
1.4. Gut microbiome of Sand-fly 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-fly and other is the amastigote form
within macrophages of infected human host [58]. There are only few
species of sand-fly which combined with Leishmania species transmit
metacyclic parasites to human host which include L. donovani—Phle-
botomusargentipes, L. major—P. papatasi,L. tropica—P. 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 flies these
microbiota affects the ability of insects as vector for the disease [62,63].
2. Characterization of gut microbiome of the sand-fly
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 affecting 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-fly 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-fly 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 identified. These
species varied based on the source of the insect’s 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 differed significantly in micro-
biome of infected sand-flies 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 different
identification methods including denaturing gradient gel electrophor-
esis (DGGE), bacterial culture have also identified 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
84
or Colombia identified 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 identified
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
fly 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-fly 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 field-collected sand –flies which might be
due to difference in food intake. P. perniciosus midgut, contained bac-
teria belonging to Burkholderia genus and Stenotrophomonas maltophilia.
They also identified few bacterial species which are not characterized in
sand-fly midgut but are present in human or other mammalian midgut
like Veillonella sp.in addition to Sporosarcinakoreensis, Rhizobium pusense
and Nocardia. The difference 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-fly microbiota and Leishmania
We have discussed that Leishmania infection in midgut of sand-flyis
accompanied by gut microbiota which has predominance of bacterial
species. It has been suggested that microbiota has an important effect
on the physiology of any disease transmitting vector and also influence
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 effector 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-fly is in-
fluenced 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
verified 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-fly is dependent on the microbiota of the
vector’s midgut. Study carried out by Kelly et al. [64]first characterized
the phylogenies of bacteria present within the midgut of three cate-
gories of sand-flyL. longipalpis i.e, sugar-fed, blood-fed and L.infantum-
infected. This observation was also accompanied by observing the effect
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 affected 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-fly 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 affect the Leishmania infection in
the sand-fly midgut [66]. The importance of exploring the area of sand-
fly 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 effect 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-
nificantly 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 significantly 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-
nificantly higher than in BALB/c mice.
When cytokine profile 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-inflammatory ef-
fects on the host. This profile shows an extensive and strong co-relation
between immune response and microbiota in self-healing mice which
was different from non-healing mice which might explain the difference
in two animal models for Leishmania infectivity. Since above section
has shown the importance of sand-fly microbiota in Leishmania infec-
tion, we focused on this and found that recently a group has given a
fundamental role of sand-fly 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-fly leads to activation of the neutrophil inflammasome
of mouse and further results in a rapid production of interleukin-1β(IL-
1β), which sustains neutrophil infiltration. Due to these neutrophils
Leishmania donovani parasites are shielded and this helps in promoting
infection of macrophages post transmission. Impairing the sand-fly
microbiota by antibiotic treatment affects the infection of Leishmania
donovani. This data gave a new insight that sand-fly midgut microbiota
can not only affect 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-specific 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-specific T cells which had the ability to
cross-react with LACK and that express a memory/effector phenotype.
This could have been the reason for secretion of IL-4 shortly after in-
fection by LACK-specificT cells.The group incubated five different
LACK-specific 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-specificT cells is due to microbial Ags,
Theseare among very few studies which has shown the effect on sys-
temic Ag-specific immune responses mediated by intestinal flora 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
85
the indigenous intestinal flora [75].
4. Metabolites and microbiota interplay in Leishmaniasis
Metabolic profiling 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
specifically differentiation 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 profiling 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 microflora 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
1
H NMR.
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-deficient 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-
filing, peripheral cytokines and faecal bacterial constituents were ana-
lysed [74]. The study identified a strong co-relation between im-
munological response, metabolic profile 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 find
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-fly vec-
tors, skin manifestation of disease will be quite challenging (Fig. 1).
Community level modularity in different 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
86
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 scientific questions to ponder upon in future.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
We are thankful for the intramural support, provided by Department
of Biotechnology, Ministry of Science and Technology, Government of
India. PM acknowledges her financial support from Council of Scientific
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:https://doi.org/10.1016/j.imlet.2019.10.004.
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