The small RNA chaperone Hfq is required for the virulence of Yersinia pseudotuberculosis.
ABSTRACT Bacterial small, noncoding RNAs (sRNAs) participate in the posttranscriptional regulation of gene expression, often by affecting protein translation, transcript stability, and/or protein activity. For proper function, many sRNAs rely on the chaperone Hfq, which mediates the interaction of the sRNA with its target mRNA. Recent studies have demonstrated that Hfq contributes to the pathogenesis of a number of bacterial species, suggesting that sRNAs play an essential role in the regulation of virulence. The enteric pathogen Yersinia pseudotuberculosis causes the disease yersiniosis. Here we show that Hfq is required by Y. pseudotuberculosis to cause mortality in an intragastric mouse model of infection, and a strain lacking Hfq is attenuated 1,000-fold compared to the wild type. Hfq is also required for virulence through the intraperitoneal route of infection and for persistence of the bacterium in the Peyer's patches, mesenteric lymph nodes, and spleen, suggesting a role for Hfq in systemic infection. Furthermore, the Deltahfq mutant of Y. pseudotuberculosis is hypermotile and displays increased production of a biosurfactant-like substance, reduced intracellular survival in macrophages, and decreased production of type III secretion effector proteins. Together, these data demonstrate that Hfq plays a critical role in the virulence of Y. pseudotuberculosis by participating in the regulation of multiple steps in the pathogenic process and further highlight the unique role of Hfq in the virulence of individual pathogens.
- SourceAvailable from: Petra Dersch[Show abstract] [Hide abstract]
ABSTRACT: Deciphering the principles how pathogenic bacteria adapt their metabolism to a specific host microenvironment is critical for understanding bacterial pathogenesis. The enteric pathogenic Yersinia species Yersinia pseudotuberculosis and Yersinia enterocolitica and the causative agent of plague, Yersinia pestis, are able to survive in a large variety of environmental reservoirs (e.g., soil, plants, insects) as well as warm-blooded animals (e.g., rodents, pigs, humans) with a particular preference for lymphatic tissues. In order to manage rapidly changing environmental conditions and interbacterial competition, Yersinia senses the nutritional composition during the course of an infection by special molecular devices, integrates this information and adapts its metabolism accordingly. In addition, nutrient availability has an impact on expression of virulence genes in response to C-sources, demonstrating a tight link between the pathogenicity of yersiniae and utilization of nutrients. Recent studies revealed that global regulatory factors such as the cAMP receptor protein (Crp) and the carbon storage regulator (Csr) system are part of a large network of transcriptional and posttranscriptional control strategies adjusting metabolic changes and virulence in response to temperature, ion and nutrient availability. Gained knowledge about the specific metabolic requirements and the correlation between metabolic and virulence gene expression that enable efficient host colonization led to the identification of new potential antimicrobial targets.Frontiers in Cellular and Infection Microbiology 10/2014; 4:146. · 2.62 Impact Factor
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ABSTRACT: Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.PLoS ONE 09/2014; 9(9):e107002. · 3.53 Impact Factor
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ABSTRACT: Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.PLoS Pathogens 06/2014; 10(6):e1004194. · 8.06 Impact Factor
INFECTION AND IMMUNITY, May 2010, p. 2034–2044
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 5
The Small RNA Chaperone Hfq Is Required for the
Virulence of Yersinia pseudotuberculosis?
Chelsea A. Schiano, Lauren E. Bellows, and Wyndham W. Lathem*
Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611
Received 12 September 2009/Returned for modification 19 October 2009/Accepted 1 March 2010
Bacterial small, noncoding RNAs (sRNAs) participate in the posttranscriptional regulation of gene expres-
sion, often by affecting protein translation, transcript stability, and/or protein activity. For proper function,
many sRNAs rely on the chaperone Hfq, which mediates the interaction of the sRNA with its target mRNA.
Recent studies have demonstrated that Hfq contributes to the pathogenesis of a number of bacterial species,
suggesting that sRNAs play an essential role in the regulation of virulence. The enteric pathogen Yersinia
pseudotuberculosis causes the disease yersiniosis. Here we show that Hfq is required by Y. pseudotuberculosis to
cause mortality in an intragastric mouse model of infection, and a strain lacking Hfq is attenuated 1,000-fold
compared to the wild type. Hfq is also required for virulence through the intraperitoneal route of infection and
for persistence of the bacterium in the Peyer’s patches, mesenteric lymph nodes, and spleen, suggesting a role
for Hfq in systemic infection. Furthermore, the ?hfq mutant of Y. pseudotuberculosis is hypermotile and displays
increased production of a biosurfactant-like substance, reduced intracellular survival in macrophages, and
decreased production of type III secretion effector proteins. Together, these data demonstrate that Hfq plays
a critical role in the virulence of Y. pseudotuberculosis by participating in the regulation of multiple steps in the
pathogenic process and further highlight the unique role of Hfq in the virulence of individual pathogens.
Small, noncoding RNAs (sRNAs) are integral components
of posttranscriptionally based regulation of protein synthesis in
prokaryotes and have been implicated in the control of quo-
rum sensing, stress response, virulence factor production, and
the regulation of outer membrane proteins (1, 7, 8, 21). Unlike
microRNAs in eukaryotes, sRNAs are often encoded in inter-
genic regions, transcribed directly from their own promoters,
and unprocessed and contain Rho-independent terminators
(34). sRNAs directly bind to their target mRNAs, and these
interactions can result in the up- or downregulation of protein
synthesis (27). For example, an sRNA molecule can bind to a
target mRNA and block the ribosome binding site or enhance
RNase E-based degradation of transcripts to inhibit transla-
tion, such as is seen with MicA-based negative regulation of
ompA in Escherichia coli (47). Conversely, an sRNA can bind
in such a way as to relieve a hairpin structure in the 5? un-
translated region of an mRNA. This exposes the ribosome
binding site to enhance translation, as has been demonstrated
in the regulation of rpoS by the sRNA DsrA (27, 43).
The Hfq protein was first identified as a host bacterial factor
required for the synthesis of bacteriophage Q? RNA (13). It is
now known that Hfq is a small (102 amino acids in E. coli, 101
amino acids in Yersinia spp.), conserved RNA chaperone pro-
tein present in many bacterial species that binds to and regu-
lates the stability of bacterial mRNA transcripts (22, 46, 50).
Furthermore, Hfq also binds to many sRNAs and enhances the
RNA-RNA interaction between these sRNAs and their
mRNA targets (35, 48, 54).
Recent studies have highlighted the contributions of Hfq
and sRNAs to bacterial pathogenesis. It has been shown that
Hfq is critical to the virulence of a number of pathogens,
including Francisella tularensis, Listeria monocytogenes, Neisse-
ria meningitidis, Salmonella enterica, and uropathogenic E. coli
(9, 12, 26, 32, 40, 42). Given the pleiotropic nature of Hfq, it is
not surprising that defects have been observed in growth under
oxidative stress and high salt and in the presence of antimicro-
bial peptides; defects in quorum sensing, host cell invasion, and
other virulence determinants have also been observed (12, 28,
42). Interestingly, the effects of Hfq seem to be unique in each
bacterial species. For example, the growth and survival inside
host cells of S. enterica and Brucella abortus, but not L. mono-
cytogenes and F. tularensis, are reduced in the absence of Hfq
(9, 32, 40, 42). In addition, regulation of species-specific factors
is often dependent on Hfq, such as the heat-stable enterotoxin
Yst of Yersinia enterocolitica and the SPI-1 regulator HilD of
Salmonella (36, 39). One common feature of Hfq among most
bacterial pathogens examined thus far is a reduction of viru-
lence in the relevant animal model in the absence of Hfq,
which supports its critical role in pathogenesis (9, 12, 26, 32,
The aim of the current work is to understand the contribution
of Hfq to the pathogenesis of Yersinia pseudotuberculosis, which is
a Gram-negative bacterium that causes yersiniosis, a generally
mild gastrointestinal disease in humans. Y. pseudotuberculosis is
very closely related to Yersinia pestis, the causative agent of plague
(6, 53). Yersiniosis caused by Y. pseudotuberculosis is character-
ized by ileitis, mesenteric lymphadenitis, fever, and diarrhea (31,
37, 41), and the presence of previous medical conditions can
increase the severity of the disease (49). Y. pseudotuberculosis is
transmitted through the fecal-oral route, and it has been shown in
mice that colonization of the Peyer’s patches occurs shortly after
the bacteria enter the intestinal lumen (30). From here dissemi-
nation to the blood, spleen, liver, and other organs can occur (3).
* Corresponding author. Mailing address: Department of Microbi-
ology-Immunology, Feinberg School of Medicine, Northwestern Uni-
versity, 303 E. Chicago Avenue, Chicago, IL 60611. Phone: (312)
503-2252. Fax: (312) 503-9594. E-mail: email@example.com.
?Published ahead of print on 15 March 2010.
The potential for Hfq to interact with multiple mRNA tar-
gets suggests that this protein may play a role in a number of
processes important to the virulence of Y. pseudotuberculosis.
Recent work has demonstrated that Hfq contributes to the
pathogenesis of Y. pestis in the mouse models of bubonic and
septicemic plague. In the absence of Hfq, Y. pestis shows a
reduced ability to colonize the spleen and liver and displays
defects in both intracellular survival and growth in vitro under
a number of stress-inducing conditions (14). Here we demon-
strate that Y. pseudotuberculosis is also attenuated for virulence
in the absence of Hfq and that Hfq participates in the regula-
tion of motility, intracellular survival, and production of type
III effectors in this bacterium.
MATERIALS AND METHODS
Reagents, bacterial strains, and growth conditions. All reagents were pur-
chased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Bacterial
strains and plasmids used in this study are listed in Table 1. Oligonucleotide
sequences are listed in Table 2. Y. pseudotuberculosis strain IP 32953 (designated
PAN29) (6), its derivatives, and all other Yersinia strains were routinely grown at
26°C in liquid brain heart infusion (BHI) broth (Difco) or on BHI agar unless
otherwise noted. E. coli strains were grown at 37°C in Luria-Bertani (LB) broth
or on LB agar. When necessary, these media were supplemented with kanamycin
(50 ?g/ml), ampicillin (100 ?g/ml), or Irgasan (2 ?g/ml). Mutants were evaluated
for the presence of key virulence loci (yopT, yopH, hmsR, and psn) by PCR.
Construction of mutant strains. A Y. pseudotuberculosis ?hfq strain was gen-
erated by homologous recombination. A 500-bp region upstream of the coding
sequence for hfq was PCR amplified from Y. pseudotuberculosis IP 32953 using
primers hfq 5?-493 Bam and hfq 3?-1 Spe, and a 500-bp region downstream of the
coding sequence was PCR amplified using primers hfq 5??1 Spe and hfq 3??500
Not. These fragments were cloned into pSR47S, which carries a kanamycin
resistance cassette and sacB (33). Clones were confirmed by DNA sequencing.
The resulting plasmid pWL302 was introduced into Y. pseudotuberculosis IP
32953 by mating. Transconjugants were plated on BHI plus kanamycin plus
Irgasan. Two kanamycin-sensitive ?hfq mutants were subsequently selected for
by passage on BHI plus 5% sucrose agar and confirmed by PCR. These mutant
strains are designated PAN38 and PAN39, respectively. The gene for Hfq was
deleted from strain Y. pseudotuberculosis 32777 as above, with wild-type and ?hfq
strains designated PAN36 and PAN40, respectively.
A deletion of the flgE gene was created in Y. pseudotuberculosis and the Y.
pseudotuberculosis ?hfq strain by using the same homologous recombination
technique as above. The up- and downstream sequences were PCR amplified
from Y. pseudotuberculosis with primers flgE 5?-501 Bam and flgE 3?-37 Spe and
primers flgE 5??1 Spe and flgE 3??498 Not, respectively. These strains are
designated PAN154 and PAN155, respectively.
The pYV plasmid, which carries the genes for the type III secretion system
(T3SS) and effectors, was cured from Y. pseudotuberculosis IP 32953 wild type
and the ?hfq mutant by growth at 37°C on BHI plates containing magnesium
chloride and sodium oxalate (MOX), as previously described (19). The loss of
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmid
Strain designation or
Genotype and/or characteristics
Y. pseudotuberculosis strains
IP 32953 ?hfq
IP 32953 ?flgE
IP 32953 ?hfq ?flgE
IP 32953 pYV?
IP 32953 ?hfq pYV?
IP 32953 ?hfq?phfq
IP 32953 ?hfq
IP 32953 ?hfq?phfq
pYV?, lacks T3SS
?hfq pYV?, lacks T3SS
?hfq hfq complemented on multicopy plasmid
?hfq hfq complemented on multicopy plasmid
?hfq hfq complemented on multicopy plasmid
Homologous recombination vector, sacB counterselection
500 bp up- and downstream of hfq coding region cloned into pSR47S
500 bp up- and downstream of flgE coding region cloned into pSR47S
C-terminal His tag coding sequence, T7 promoter
pET24a(?) with Yersinia hfq C-terminal His tag for overexpression
pUC-ori LacZ? placTOPO cloning site
hfq gene and 1-kb upstream sequence inserted into pCR-Blunt II-TOPO
TABLE 2. Oligonucleotides used in this study
hfq 5?-493 Bam...............CGGGATCCCGGGTGAAACCTTACCTTACCG
hfq 3?-1 Spe ....................GGACTAGTTCTATATTTTCCTTATTTGCTTG
hfq 5??1 Spe ..................GGACTAGTAGCCCATTGCTGGTCGACCATG
hfq 3??500 Not..............ATAAGAATGCGGCCGCGGTCGCGATATGACG
flgE 5?-501 Bam .............GGATCCCATGGCAAAGCTGCTCAAGAGC
flgE 3?-37 Spe.................ACTAGTGATATTGA CCGTGCGGGCTAG
flgE 5??1 Spe.................ACTAGTTGGATAAGCTTCTGTATACCGCC
flgE 3??498 Not.............GCGGCCGCGCGGGGATATCCACCAGTTTG
hfq 5?-1018 Bam.............CGGGATCCTTATTATG GGGCCAACTGCTTC
hfq 3?306 Eco.................GGAATTCTTATTCAGCGTCATCACTGTC CTGC
hfq 5?1 Nde.....................GGAATTCCATATGGCTAAGGGGCAATCTTTGC
hfq 3?303 Xho.................CCGCTCGAGTTCAGCGTCATCACTGTCCTG
hflX 5?1100 .....................ACTTTGAATTGCGCTTGCCTCCTC
hflX 3?1215 .....................TCTAACCACCATACCGACATTCCC
VOL. 78, 2010ROLE OF Hfq IN Y. PSEUDOTUBERCULOSIS VIRULENCE2035
pYV was verified by PCR and by the loss of growth restriction at 37°C in the
absence of calcium (15). These mutant strains are designated PAN100 and
Construction of complementing plasmid phfq. The coding region and 1,018 bp
upstream of the transcriptional start site for hfq were PCR amplified from Y.
pseudotuberculosis using primers hfq 5?-1018 Bam and hfq 3?306 Eco. This
product was inserted into the plasmid vector pCR-Blunt II-TOPO (Invitrogen),
and the sequence was verified. The plasmid, called phfq, was transformed into
both Y. pseudotuberculosis IP 32953 ?hfq strains and the 32777 ?hfq strain by
electroporation, and the strains were designated PAN136, PAN181, and
Antibody production and immunoblot analysis. The coding sequence for Hfq
was PCR amplified from Yersinia using primers hfq 5?1 Nde and hfq 3?303 Xho
and cloned into plasmid pET24a(?), which contains a C-terminal hexahistidine
tag. The His-tagged Hfq protein was expressed in E. coli BL21(DE3) and puri-
fied under native conditions according to the Qiagen Expressionist protocol, and
polyclonal anti-Hfq antibodies were raised in rabbits (Covance).
For immunoblotanalysis, Y.pseudotuberculosis
?hfq?phfq strains (PAN29, PAN38, and PAN136, respectively) were grown
overnight in BHI at 26°C; equivalent units of optical density at 620 nm (OD620)
were taken from each culture, resuspended in sample buffer (10% glycerol, 100
mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.02 mg/ml bromophe-
nol blue, 5% ?-mercaptoethanol), and separated by SDS-PAGE. Proteins were
transferred to nitrocellulose membranes for analysis of Hfq expression with the
anti-Hfq antibody by immunoblotting.
Growth curves. Y. pseudotuberculosis wild-type, ?hfq, and ?hfq?phfq strains
(PAN29, PAN38, and PAN136, respectively) were cultured overnight in BHI at
26°C and then subcultured at an OD620of 0.1 in 10 ml of BHI, BHI plus 2.5 mM
CaCl2, M9, or M9 plus 2.5 mM CaCl2. Cultures were incubated with shaking at
250 rpm in 125-ml Erlenmeyer flasks at 26°C or 37°C for 12 h. Optical density was
measured at 620 nm.
Animal infections. All procedures involving animals were carried out in com-
pliance with protocols approved by the Northwestern University institutional
animal care and use committee. Eight-week-old female BALB/c mice were
purchased from Harlan Laboratories and allowed to acclimate to the animal
facility for 5 to 7 days prior to infection. To prepare the inocula, Y. pseudotu-
berculosis wild-type, ?hfq, and pYV?strains (PAN29, PAN38, and PAN100,
respectively) were cultured overnight in BHI at 26°C, diluted to an OD620of 0.1,
and incubated at 26°C with shaking to an OD620of 0.6. The cells were harvested
by centrifugation, washed once with sterile phosphate-buffered saline (PBS), and
diluted to the appropriate OD620in PBS. Groups of 10 mice were inoculated
intragastrically using a 22-gauge feeding needle with approximately 107CFU of
the Y. pseudotuberculosis wild-type, ?hfq, or pYV?strain and monitored for 21
days. The weights of individual mice were recorded every third day. In experi-
ments examining the kinetics of infection, groups of 5 mice were infected as
above and sacrificed at various times postinoculation. CFU per gram of tissue
were determined in the spleen, visible Peyer’s patches, tissue inclusive of mes-
enteric lymph nodes, and the small intestine by homogenizing organs in PBS and
plating them on Yersinia selective agar (Difco). Mice with CFU counts in any
organ or with any recorded weight loss were included in the analysis. For the
dose-response survival curve, mice were anesthetized with a mixture of ketamine
(100 mg/kg of body weight) and xylazine (10 mg/kg) in PBS given intraperito-
neally immediately prior to intragastric inoculation with either 10-fold-increasing
doses of the Y. pseudotuberculosis ?hfq strain (103to 107CFU) or 103CFU of the
wild-type strain and monitored for 21 days. For intraperitoneal infections, mice
were injected using a 28-gauge needle with approximately 103CFU of the Y.
pseudotuberculosis wild-type, ?hfq, or pYV?strain and monitored for 21 days.
Gentamicin protection assays. J774 murine macrophage-like cells were rou-
tinely cultured in Dulbecco’s modified Eagle’s medium (Cellgro; Mediatech)
plus 10% heat-inactivated fetal calf serum (HyClone) and penicillin-streptomy-
cin (100 ?g/ml) (Cellgro; Mediatech) at 37°C in a 5% CO2environment. Cells
(5 ? 105) were seeded into 24-well plates 16 to 18 h prior to use. Adherent cells
were washed with PBS and incubated for 1 h with standard media lacking
penicillin-streptomycin (binding buffer). Y. pseudotuberculosis wild-type, ?hfq,
and ?hfq?phfq strains were cultured overnight in BHI at 26°C, washed with PBS,
diluted to the appropriate CFU/ml in binding buffer (multiplicity of infection
[MOI] of 10), and added to the J774 cells. The bacteria were incubated with host
cells for 1 h. Cells were then washed five times in PBS and incubated for 30 min
with binding buffer or binding buffer supplemented with 100 ?g/ml gentamicin
(Invitrogen). After 30 min cells were washed five times with PBS and lysed with
0.1% Triton X-100 in water, and serial dilutions were plated onto BHI plates to
determine the number of host cell-associated bacterial cells. The inocula were
also plated to calculate percent association relative to inocula. Alternatively,
after 30 min of incubation with 100 ?g/ml gentamicin, cells were washed once
with PBS and incubated with binding buffer supplemented with 10 ?g/ml gen-
tamicin for 2 and 4 h. Cells were then washed and lysed as above. The experi-
ments were performed in triplicate and with at least 3 independent biological
Hydrogen peroxide killing assay. Y. pseudotuberculosis wild-type, ?hfq, and
?hfq?phfq strains (PAN29, PAN38, and PAN136, respectively) were grown
overnight in BHI at 26°C and subcultured to an OD620of 0.1. Bacteria were
grown to mid-log phase and then diluted 1:10 in BHI. Hydrogen peroxide diluted
in water or water alone was added to the bacteria to a concentration of 100 mM.
All samples were incubated at 26°C in a roller drum, and aliquots were taken at
10 and 30 min posttreatment. Serial dilutions were plated onto BHI plates to
determine the CFU/ml in the treated versus untreated samples. Experiments
were performed in triplicate and with three independent biologic replicates.
Motility and biosurfactant assays. Colonies of Y. pseudotuberculosis wild-type,
?hfq, ?hfq?phfq, ?flgE, and ?hfq ?flgE strains (PAN29, PAN38, PAN136,
PAN154, and PAN155, respectively) were cultured overnight in BHI at 26°C.
Aliquots (2 ?l) of each were spotted onto soft agar motility plates (1.0% tryp-
tone, 0.5% NaCl, 0.3% agar) and incubated at 22°C or 37°C, and at various times
photographs were taken using the Gel Doc XR System (Bio-Rad). Aliquots (2
?l) of water were spotted inside and outside the border of the refractive com-
pound to assess surface tension. Images of the released compound were taken
using a Cannon 60D camera with a 100 mM F macrolens 2 days after the bacteria
were spotted onto plates.
qRT-PCR. Overnight cultures of Y. pseudotuberculosis wild-type and ?hfq
strains (PAN29 and PAN38, respectively) were grown in triplicate and diluted to
an OD620of 0.1 in 22 ml BHI plus MOX. For the flhC, flgA, fliC, and hflX genes,
cultures were grown at 26°C for 6 h. For the yopE, yopH, yopJ, and yopT genes,
cultures were grown at 26°C for 1 h and then shifted to 37°C for 3 h. Five OD
units were removed and added to RNAprotect bacterial reagent (Qiagen). RNA
was isolated using the RiboPure bacterial kit (Ambion) and treated with DNase,
and cDNA was synthesized with SuperScript II reverse transcriptase (Invitro-
gen). Quantitative reverse transcriptase PCR (qRT-PCR) was performed with
the SYBR green dye in an iCycler thermocycler (Bio-Rad). The calculated
threshold cycle (CT) was normalized to the CTof the gyrB gene from the same
cDNA sample before calculation of the fold changes using the ??CTmethod (2).
Type III secretion assays. Y. pseudotuberculosis wild-type, ?hfq, and pYV?
strains (PAN29, PAN38, and PAN100, respectively) were diluted to an OD620of
0.1 in BHI plus MOX and cultured at 26°C for 1 h followed by 37°C for 3 h.
Bacteria were centrifuged, and equivalent OD units of culture supernatants were
harvested, filtered, and precipitated by the addition of trichloroacetic acid to
10%. Precipitated proteins were resuspended in equal volumes of 1 M Tris (pH
9.0) and sample buffer, separated by SDS-PAGE, and transferred to nitrocellu-
lose or stained with Coomassie brilliant blue. For normalization purposes, 0.2
OD units of bacterial cultures was harvested and separated by SDS-PAGE.
Bacterial cell pellets were washed three times with PBS, resuspended in PBS plus
lysozyme (0.5 mg/ml), and incubated on ice for 30 min. Cells were then sonicated,
and cellular debris was removed by centrifugation. Whole-cell lysates (50 ?g)
were separated by SDS-PAGE and transferred to nitrocellulose. Immunoblot
analyses were performed using antibodies to YopE, YopH, YopJ, YopT, and
RpoA (as a loading control).
Statistical analysis. All experiments were performed two or more times, un-
less otherwise noted. Student’s unpaired t test was used to compare wild-type,
?hfq, and ?hfq?phfq strains in growth curves, in the gentamicin protection assay,
and in the hydrogen peroxide killing assay and to compare mouse weights during
intragastric infection. For qRT-PCR experiments, significance was calculated by
the Wilcoxon signed-rank test. For kinetics experiments, significant differences in
CFU/organ were determined by the Mann-Whitney U test. In all cases, a P value
of less than 0.05 was considered significant.
Deletion of hfq from Y. pseudotuberculosis and growth in vitro.
In order to examine the role of Hfq in the virulence of Y.
pseudotuberculosis, we generated an unmarked isogenic mutant
lacking the entire Hfq coding sequence. The loss of Hfq was
verified by immunoblot analysis of overnight cultures of Y.
pseudotuberculosis (data not shown). In addition, we confirmed
the absence of polar effects on hflX, the gene immediately
downstream of hfq, by qRT-PCR (data not shown). We then
2036 SCHIANO ET AL.INFECT. IMMUN.
generated a plasmid-based complementing clone of hfq. Pro-
duction of Hfq protein was detected in the complemented
mutant, and Hfq appears to be noticeably overproduced in this
strain compared to wild type, which is likely due to the multi-
copy nature of the plasmid (data not shown).
The absence of Hfq in some bacterial species has been
associated with growth defects in various types of liquid media
(12, 42, 50). In order to determine the effects of Hfq on the
growth of Y. pseudotuberculosis, the wild-type, ?hfq, and
?hfq?phfq strains (PAN29, PAN38, and PAN136, respec-
tively) were cultured in BHI (rich) broth at 26°C, 37°C, and
37°C plus 2.5 mM CaCl2. At 26°C there is no effect of Hfq on
growth (Fig. 1), and overnight cultures of wild-type and ?hfq
strains routinely reach the same density (data not shown). At
37°C, the loss of Hfq results in a slight, though not statistically
significant, growth defect (Fig. 1). At 37°C in the presence of
calcium, however, ?hfq bacteria exhibit a statistically signifi-
cant growth defect during both exponential phase (P ? 0.0419)
and stationary phase (P ? 0.0003) (Fig. 1). In particular, at
37°C with calcium Y. pseudotuberculosis has a slower doubling
time in the absence of Hfq (1.35 h versus 3.85 h), and the ?hfq
culture never reaches the optical density of the wild type. This
defect is partly ameliorated in the complemented strain in
stationary phase (P ? 0.0061) (Fig. 1). The effects of Hfq on
the growth of Y. pseudotuberculosis in a nutrient-limiting envi-
ronment were also examined. Wild-type, ?hfq, and ?hfq?phfq
bacteria were cultured in M9 medium as above (data not
shown). As expected, all three strains reached a lower station-
ary-phase optical density than in the rich media; however, the
observed trends remained the same for growth in M9 as in
BHI. No significant growth defect was seen in the absence of
Hfq at 26°C or at 37°C in the absence of CaCl2(data not
shown). A significant difference was seen during exponential
phase when the Y. pseudotuberculosis ?hfq strain was cultured
at 37°C with 2.5 mM CaCl2(P ? 0.0237) (data not shown).
The Y. pseudotuberculosis ?hfq strain is attenuated for viru-
lence in an intragastric mouse model of infection. To deter-
mine if Hfq is required for the pathogenesis of Y. pseudotuber-
culosis in a model that mimics a natural route of infection,
9-week-old female BALB/c mice were infected intragastrically
with 106or 107CFU of wild-type, ?hfq, or pYV?bacteria
(PAN29, PAN38, and PAN100, respectively) and monitored
for 21 days. When given 106CFU, 100% of mice infected with
the ?hfq strain survived, but only 30% survived when infected
with the wild-type Y. pseudotuberculosis (data not shown).
When inoculated with 107CFU, 90% of mice infected with the
wild-type strain died by day 15, while all mice infected with the
?hfq strain survived for the duration of the experiment (Fig.
2a). As expected, all mice infected with the pYV-cured strain
survived, since this strain lacks the T3SS and effectors that are
essential for Yersinia virulence. We also determined the weight
of each mouse every 3 days, as the loss of body weight is
indicative of a symptomatic infection. Mice infected with wild-
type Y. pseudotuberculosis suffered significant weight loss prior
to succumbing to the infection (Fig. 2b). Mice infected with the
?hfq strain initially displayed mild, but not statistically signif-
icant, weight loss but eventually recovered, while those in-
fected with the pYV?strain never lost weight (Fig. 2b).
In order to determine if there is a dose at which Y. pseudo-
tuberculosis deleted for hfq affects the survival of mice, we
adapted a model in which the infectious dose can be lowered
from 107CFU to 103CFU using a ketamine-xylazine cocktail
to anesthetize the mice prior to intragastric inoculation. This
model is useful because doses of bacteria can be administered
many times higher than the 100% lethal dose (LD100) for
wild-type Y. pseudotuberculosis without risking septic shock
FIG. 1. Growth of Y. pseudotuberculosis ?hfq strain in rich media. Y. pseudotuberculosis wild-type, ?hfq, and ?hfq?phfq strains were cultured
in BHI broth at 26°C, 37°C, and 37°C plus 2.5 mM CaCl2, and the OD620of each culture was measured over the course of the growth curve. Each
graph represents the mean of three independent biological replicates grown on three different days. The error bars represent the standard deviation
of the optical density at each time point. Significance was calculated by Student’s unpaired t test at 4 and 12 h (*, P ? 0.0419;***, P ? 0.0003).
VOL. 78, 2010ROLE OF Hfq IN Y. PSEUDOTUBERCULOSIS VIRULENCE 2037
that would likely occur with the unanesthetized infection
model (i.e., CFU approaching 1011) (24, 51). Ketamine-xyla-
zine has been shown to induce a passing immunosuppressive
state in rat gut mucosal homogenates by reducing inducible
nitric oxide synthase (iNOS) and tumor necrosis factor alpha
(TNF-?) production that lasts for 1 to 3 h after treatment (17,
45), which we hypothesize allows the bacteria to cause an
infection at a lower dose. This hypothesis is supported by the
fact that ketamine treatment allows for an intragastric Vibrio
cholerae infection of adult mice, which is not possible in the
absence of the anesthetic (38). Importantly, mice treated with
ketamine-xylazine and infected with 103CFU of avirulent,
pYV?Y. pseudotuberculosis (lacking the T3SS plasmid) do not
succumb to the infection and do not display any weight loss in
this model (data not shown). Mice were injected intraperito-
neally with a ketamine-xylazine cocktail immediately prior to
infection with 103CFU of wild-type Y. pseudotuberculosis or
10-fold-increasing doses of the Y. pseudotuberculosis ?hfq
strain from 103to 107CFU. All ketamine-xylazine-treated
mice infected with wild-type bacteria succumbed to the infec-
tion by 8 days postinoculation, while none of the ketamine-
xylazine-treated mice infected with 103to 106CFU of the ?hfq
strain died (Fig. 3). None of the ketamine-xylazine-treated
mice infected with the highest dose of the Y. pseudotuberculosis
?hfq strain survived beyond day 3 (Fig. 3). These data indicate
that the Y. pseudotuberculosis ?hfq strain is at least 1,000-fold
less virulent than the wild-type strain under these conditions.
The defect in virulence of Y. pseudotuberculosis lacking Hfq
may be due to a reduced ability of the bacteria to colonize the
small intestine, to disseminate to other organs, or to persist in
these organs. In order to evaluate the role of Hfq in bacterial
colonization of these organs, unanesthetized mice were in-
fected intragastrically with 107CFU of the Y. pseudotubercu-
losis wild-type or ?hfq strain (PAN29 and PAN38, respec-
tively). Mice were sacrificed 2, 4, 6, and 9 days postinoculation,
and the small intestine, Peyer’s patches, mesenteric lymph
nodes, and spleen were removed, homogenized, and plated to
determine the bacterial load in each organ. No significant
difference was observed in the ability of the Y. pseudotubercu-
losis ?hfq strain to colonize the small intestine on day 2 post-
inoculation compared to the wild type, nor did the bacterial
burden in the small intestine differ between wild-type- and
?hfq strain-infected mice on day 4 or 6 (Fig. 4a). However, a
significant difference in CFU/g was seen on day 9 in the small
intestine, with a trend toward clearance of the ?hfq strain
occurring on or by this day (Fig. 4a). Similarly, there was no
significant difference in the bacterial load in the Peyer’s
patches on day 4 or 6 between wild-type- and ?hfq strain-
infected mice (Fig. 4b). A significant difference in bacterial
load was seen in the Peyer’s patches on days 2 and 9, however
(Fig. 4B). In addition, the wild-type and ?hfq strains were able
to disseminate to the mesenteric lymph nodes and spleen
equally well on day 2 (Fig. 4c and d). A significant difference in
the bacterial load in the mesenteric lymph nodes and spleen
was observed on days 4, 6, and 9, indicating that in the absence
of Hfq, Y. pseudotuberculosis may be diminished in its ability to
survive or replicate in lymphoid organs (Fig. 4b to d).
Hfq is required for the virulence of Y. pseudotuberculosis in a
systemic model of infection. In order to further investigate if Y.
FIG. 2. Survival of mice inoculated intragastrically with the Y.
pseudotuberculosis ?hfq strain. Groups of 10 mice were inoculated via
oral gavage with the Y. pseudotuberculosis wild-type, ?hfq, or pYV?
strain (107CFU) and monitored for 21 days. (a) Survival of infected
mice over 21 days. (b) Body weight of infected mice over 21 days. The
plot shows median weight, indicated by a solid line; a box represents
the 25th and 75th percentiles, and whiskers represent the range. Sig-
nificance was calculated by Student’s unpaired t test.
FIG. 3. Survival of mice inoculated with increasing doses of the Y.
pseudotuberculosis ?hfq strain. Groups of 5 mice were injected intra-
peritoneally with a ketamine-xylazine cocktail immediately prior to
intragastric inoculation with Y. pseudotuberculosis (103CFU) or 10-
fold-increasing doses of the Y. pseudotuberculosis ?hfq strain (103to
107CFU). Data are representative of 2 independent experiments.
2038 SCHIANO ET AL.INFECT. IMMUN.
pseudotuberculosis requires Hfq only in the initial stages of
colonization or if it is also necessary for a systemic infection,
mice were infected via the intraperitoneal route with 103CFU
of the Y. pseudotuberculosis wild-type, ?hfq, or pYV?strain
(PAN29, PAN38, and PAN100, respectively) and survival was
monitored for 21 days. This allowed the infection to bypass the
step where Y. pseudotuberculosis colonizes the small intestine.
Of the wild-type-infected mice, only 10% survived for the du-
ration of the experiment, while in the ?hfq and pYV?strain-
infected groups, all mice survived for 21 days (Fig. 5). This
further suggests that Hfq is critical for the virulence of Y.
pseudotuberculosis beyond the initial colonization steps and is
required during a systemic infection.
The Y. pseudotuberculosis ?hfq strain is hypermotile at 22°C.
In order to determine how the absence of Hfq could lead to
such a severe reduction in virulence, we examined key steps in
the infectious process. As motility is a significant virulence
determinant in many bacterial pathogens, we examined the
effect of Hfq on the swarming motility of Y. pseudotuberculosis
by growth on low-percentage agar plates. Overnight cultures of
the Y. pseudotuberculosis wild-type, ?hfq, or ?hfq?phfq strain
(PAN29, PAN38, and PAN136, respectively) were spotted
onto swarm plates and grown at either 22°C or 37°C for 4 days.
The absence of hfq results in increased motility at 22°C but not
at 37°C, which indicates that Hfq is essential for the negative
regulation of motility in Y. pseudotuberculosis at lower temper-
atures (Fig. 6a). To determine if hypermotility in the absence
of Hfq is mediated through changes in flagellar synthesis or
activity, we generated a deletion of the gene for the flagellar
hook protein, flgE. The deletion of flgE in both the wild-type
and ?hfq bacteria resulted in the same motility phenotype as
that of the parental strains (Fig. 6a). Furthermore, qRT-PCR
revealed that there is no significant difference in the transcript
levels of early, middle, or late flagellar genes (flhC, flgA, and
fliC) between the wild-type and mutant strains (Fig. 6b). Taken
together, these data suggest that the hypermotility of the Y.
FIG. 4. Kinetics of infection with the Y. pseudotuberculosis ?hfq strain. Mice were inoculated intragastrically with wild-type or ?hfq Y.
pseudotuberculosis (107CFU), and after 2, 4, 6, and 9 days, CFU per gram of tissue in the spleen, visible Peyer’s patches, mesenteric lymph nodes,
and small intestine were determined. Graphs show bacterial counts from 3 combined experiments. Each point represents CFU/g recovered from
a single animal (E, wild-type strain; ?, ?hfq strain). A dashed line indicates the limit of detection. A solid line indicates the median of CFU
recovered. Symbols below the limit of detection represent mice that survived but did not have detectable numbers of bacteria. Statistical
significance was calculated by the Mann-Whitney U test.
FIG. 5. Survival of mice inoculated intraperitoneally with the Y.
pseudotuberculosis ?hfq strain. Groups of 10 mice were inoculated by
intraperitoneal injection with wild-type, ?hfq, or pYV?Y. pseudotu-
berculosis (103CFU) and monitored for 21 days. Data are represen-
tative of two independent experiments; in one of the experiments, a
single mouse infected with the ?hfq strain succumbed to the infection
on day 11 (not shown).
VOL. 78, 2010ROLE OF Hfq IN Y. PSEUDOTUBERCULOSIS VIRULENCE2039
pseudotuberculosis ?hfq strain is not mediated through changes
in flagellar expression compared to wild type.
Enhanced production of a biosurfactant-like substance in
the absence of Hfq. Close examination of the low-percentage
agar plates described above show a light-refractive compound
visible in the agar surrounding the Y. pseudotuberculosis colony
that is more evident in the absence of Hfq. The diameter of the
refractive compound is larger in the absence of Hfq and is not
affected by the presence or absence of flgE (Fig. 7a). Cultures
of this material did not yield any bacterial growth, nor is this
phenotype dependent on the T3SS (data not shown). Stewart
et al. observed the production of a similar light-refractive com-
pound by Legionella pneumophila, which they identified as a
biosurfactant (44). Biosurfactants are characterized by their
ability to reduce surface tension (11); therefore, in order to
determine if the refractive compound observed here has prop-
erties of a biosurfactant, the Y. pseudotuberculosis ?hfq strain
was plated on soft agar plates as above and allowed to grow for
2 days, after which time droplets of water were spotted inside
and outside the area of refraction. The droplet of water inside
collapsed immediately, while the droplet spotted outside main-
tained its integrity, which is consistent with the reduction in
surface tension characteristic of a biosurfactant (Fig. 7b).
The loss of Hfq reduces intracellular survival of Y. pseudo-
tuberculosis in macrophage-like cells. Previous work has shown
that the intracellular survival of Y. pestis in cultured host mac-
rophage cells is defective in the absence of Hfq (14). As this
may be a critical step in pathogenesis, we examined the impact
of Hfq on the ability of Y. pseudotuberculosis to survive within
cultured macrophages. Y. pseudotuberculosis wild-type, ?hfq,
and ?hfq?phfq strains (PAN29, PAN38, and PAN136, respec-
tively) were incubated with J774 murine macrophage-like cells
for 1 h (MOI of 10) and treated with gentamicin for 30 min,
and CFU were determined. While there was a significant dif-
ference in the absolute numbers of cell-associated bacteria
between the wild-type and ?hfq strains (Fig. 8a), there was no
difference in the percentages of intracellular bacteria between
the two strains (Fig. 8b). After 2 and 4 h of gentamicin treat-
ment, however, the intracellular survival of the Y. pseudotuber-
culosis ?hfq strain was significantly decreased compared to that
of the wild type (Fig. 8b). After 2 h and 4 h the phfq comple-
menting strain is unable to restore survival to wild-type levels
(Fig. 8b). We observed the same defect in a second, indepen-
dently derived ?hfq mutant and complement of Y. pseudotu-
berculosis IP 32953 (PAN39 and PAN181, respectively), as well
as in an ?hfq mutant and complement of a different isolate of
Y. pseudotuberculosis, strain 32777 (PAN40 and PAN137, re-
spectively) (data not shown). This suggests a defect with the
FIG. 6. Motility of the Y. pseudotuberculosis ?hfq strain on semi-
solid agar. (a) Y. pseudotuberculosis wild-type, ?hfq, ?flgE, ?hfq ?flgE,
and ?hfq?phfq strains were cultured on semisolid agar plates at 22°C
or 37°C, and motility was monitored at 1 and 4 days postinoculation.
Images are representative of several experiments. (b) qRT-PCR of
flhC, flgA, and fliC transcripts. Bars represent the relative average fold
change compared to wild type for each transcript of 3 independent
FIG. 7. Production of a biosurfactant-like substance by the Y.
pseudotuberculosis ?hfq strain. (a) Bacteria were prepared as described
for Fig. 6 and cultured at 22°C for 2 days before photographs of a
light-refractive compound, visible as a clear ring surrounding the bac-
terial colony, were taken. (b) Droplets of water were spotted inside and
outside the compound on the motility plate 2 days after plating to
demonstrate the reduced surface tension. A dotted white line delin-
eates the border of the refractive compound.
2040SCHIANO ET AL.INFECT. IMMUN.
plasmid-based complementation in this assay, rather than sec-
ondary, pleiotropic mutations in ?hfq bacteria. These data
indicate that Hfq is necessary for adherence and intracellular
survival of Y. pseudotuberculosis in host macrophages.
Hfq contributes to the resistance to oxidative stress. In
order to determine if the defect in intracellular survival of Y.
pseudotuberculosis in the absence of Hfq is due to a decreased
ability of the bacteria to survive the oxidative burst, we exposed
Y. pseudotuberculosis wild-type, ?hfq, and ?hfq?phfq (PAN29,
PAN38, and PAN136, respectively) bacteria to H2O2. In the
absence of Hfq, Y. pseudotuberculosis was significantly reduced
in its ability to survive in the presence of H2O2after 10 and 30
min of treatment (Fig. 9).
Dysregulation of the T3SS in the absence of Hfq. The type
III secretion system (T3SS) is required by all pathogenic Yer-
sinia species, including Y. pseudotuberculosis, for mammalian
virulence (10). In order to determine if Hfq plays a role in the
regulation of the T3SS in Y. pseudotuberculosis, bacteria were
cultured for 3 h under secretion-inducing conditions (37°C, low
Ca2?), and the culture supernatants and cell lysates from wild-
type, ?hfq, and pYV?(PAN29, PAN38, and PAN100, respec-
tively) bacteria were analyzed for the presence and abundance
of Yop proteins. The overall protein profile was determined
using Coomassie brilliant blue staining, and levels of the effec-
tor proteins YopE, YopH, YopJ, and YopT were assessed by
immunoblotting with antibodies specific to each. The Coomas-
sie blue-stained gel revealed that Hfq does not globally affect
levels of secretion (supernatant) (Fig. 10a). In the absence of
Hfq we observed decreased levels of all four Yop proteins
tested in the cell pellets as well as the culture supernatants
(Fig. 10b). To determine if the altered amounts of Yop pro-
teins were due to changes in transcript abundance, we exam-
ined by qRT-PCR the relative mRNA levels of each Yop
between the wild-type and ?hfq strains. There was no signifi-
cant difference in the expression of any of the yop transcripts
between wild-type and ?hfq bacteria (Fig. 10c). These data
suggest that Hfq participates in the positive regulation of the
T3SS, likely at a posttranscriptional level.
This study demonstrates that the small RNA chaperone Hfq
plays a critical role in the pathogenesis of the enteric pathogen
Y. pseudotuberculosis. A mouse model of infection shows that
Hfq is required for the virulence of Y. pseudotuberculosis by a
natural route of infection (Fig. 2a), and in the absence of Hfq,
Y. pseudotuberculosis-infected mice do not succumb to the in-
fection as their wild-type-infected counterparts do, even with a
1,000-fold-higher dose of bacteria (Fig. 3). This reduction in
virulence may be due to the decreased ability of Hfq-deficient
Y. pseudotuberculosis to survive and replicate in the Peyer’s
patches, mesenteric lymph nodes, and spleen (Fig. 4b to d).
The loss of Hfq does not completely abrogate the ability of
Y. pseudotuberculosis to establish an infection in the mouse,
however, as demonstrated by the moderate weight loss of the
animals and the bacterial burden in the tissues of ?hfq strain-
infected mice (Fig. 2b and 4a to d). In other pathogens, the loss
of Hfq often results in a severe, multifold defect in the bacte-
rial load in tissues and organs compared to a wild-type infec-
tion (14, 26, 42), whereas with Y. pseudotuberculosis, this does
not appear to be the case. This suggests a unique contribution
to virulence for Hfq in Y. pseudotuberculosis. While the atten-
uation in virulence attributed to Hfq may be due to the defect
in growth at 37°C, it is also possible that Hfq contributes to the
subversion of the host innate and adaptive immune response
by Y. pseudotuberculosis. Hfq-dependent sRNAs may partici-
FIG. 8. Association of the Y. pseudotuberculosis ?hfq strain with
host cells. The Y. pseudotuberculosis wild-type, ?hfq, or ?hfq?phfq
strain was incubated with J774 murine macrophage-like cells (MOI of
10). (a) Percentage of inoculum associated with host cells. (b) Percent
intracellular bacteria after 30 min of treatment with gentamicin com-
pared to untreated cells and percent intracellular bacteria after 2 and
4 h compared to CFU after 30 min. Bars represent the mean percent-
ages, and error bars are standard errors of CFU from triplicate wells.
Statistical analysis was performed with Student’s unpaired t test. Data
are representative of 3 independent experiments.
FIG. 9. Survival in the presence of hydrogen peroxide. Y. pseudo-
tuberculosis wild-type, ?hfq, and ?hfq?phfq strains were incubated for
10 or 30 min with 100 mM H2O2. Bars represent mean percent survival
compared to untreated controls, and error bars represent standard
errors of percent survival from 3 replicates. Statistical analysis was
performed with Student’s unpaired t test. Data are representative of 3
VOL. 78, 2010 ROLE OF Hfq IN Y. PSEUDOTUBERCULOSIS VIRULENCE2041
pate in the regulation of the expression of proteins that alter
the immunogenicity of the bacterium or may affect the ability
of Y. pseudotuberculosis to express virulence factors that pre-
vent clearance by the host. Indeed, the Hfq homolog of F.
tularensis was identified as an activator of the host immune
system, supporting this possibility (16).
Alternatively, Hfq may have a role in regulating specific
virulence factors that are essential for survival or replication in
the lymphoid tissue. For instance, in the absence of Hfq the
motility of Y. pseudotuberculosis is increased compared to wild-
type bacteria (Fig. 6). While expression of the flagellar genes is
also repressed at 37°C in Y. pseudotuberculosis, the observed
hypermotility is independent of the flagellum (Fig. 6). Thus, we
expect that this form of motility would not be overridden by the
transcriptional regulation of flagellar expression (23). Non-
flagellum-based motility could play a role in mammalian infec-
tion, particularly if the expression of the sRNA(s) that re-
presses this phenomenon in wild-type bacteria in vitro is
downregulated in vivo. Our results add an additional layer of
sRNA-based regulation of motility beyond the CsrABC system
that affects FlhDC expression in Y. pseudotuberculosis (18).
This phenotype is in contrast to the decreased motility ob-
served when Hfq is deleted from Salmonella enterica serovar
Typhimurium and uropathogenic E. coli, which further dem-
onstrates that the effects of Hfq are unique to each bacterial
species (26, 42).
An unexpected result from this study is the discovery of a
biosurfactant-like substance that is released by the Y. pseudo-
tuberculosis ?hfq strain on a semisolid surface. This observa-
tion is intriguing, as biosurfactant production by Yersinia spe-
cies has not been reported. Interestingly, Y. pestis does not
produce this compound in either the presence or the absence
of Hfq (data not shown). The biosurfactant-like substance is
not produced at 37°C in our assay, which correlates with the
presence of the hypermotility phenotype occurring only at
lower temperatures and suggests that this substance could con-
tribute to the hypermotility of the Y. pseudotuberculosis ?hfq
strain at 22°C. Although the biosurfactant does not appear to
be produced at physiologic temperatures in vitro, there may be
stimuli that promote its synthesis under certain conditions dur-
ing infection. Indeed, the in vivo function of this biosurfactant-
like substance is unknown, but it could play a role during
infection, as a biosurfactant as has been implicated in the
virulence of Pseudomonas (5). The overproduction of the bio-
surfactant-like substance may contribute to the reduction in
virulence in the mouse model caused by enhanced motility, a
reduced ability to adhere to host cells, increased stimulation of
the immune response, or another function, all of which may
contribute to defects in the ability of Y. pseudotuberculosis to
persist in lymphoid organs. A database search revealed puta-
tive glycosyltransferases in the genome of Y. pseudotuberculosis
that are similar to the genes for rhlB and rhlC in Pseudomonas
aeruginosa. YPTB1978, encoding a putative glycosyltrans-
ferase, with 39% similarity to rhlB, and speE, encoding a sper-
midine synthase, with 44% similarity to rhlC, may have func-
tions that could be part of a biosurfactant synthesis pathway,
and in other bacterial species, such genes have been implicated
in changes in motility (4). This avenue requires further inves-
tigation to determine the biosynthetic pathway and biosurfac-
tant material, as well as its potential contribution to virulence.
Appropriate host-pathogen interactions involving the mac-
rophage are critical to the virulence of many bacterial species.
It is possible, then, that the attenuation of the ?hfq strain may
be explained by defects in the interaction of Y. pseudotubercu-
losis with host immune cells in the Peyer’s patches, spleen, and
mesenteric lymph nodes following dissemination from the
small intestine. In the absence of Hfq, Y. pseudotuberculosis
exhibits a significant defect in intracellular survival in macro-
phage-like cells (Fig. 8). This may be due to the reduced ability
of the Y. pseudotuberculosis ?hfq strain to withstand the oxi-
dative burst, simulated by the H2O2killing assay (Fig. 9). Our
experiments highlight the differences and unique role that Hfq
and small RNAs can play in different pathogens. The loss of
Hfq also reduces the growth and survival of S. enterica and B.
abortus within the macrophage, suggesting that Hfq contrib-
utes to the regulation of factors that are involved in intracel-
FIG. 10. Production of type III secretion effector proteins in the
absence of Hfq. Y. pseudotuberculosis wild-type, ?hfq, and pYV?
strains were cultured in BHI broth for 3 h under Yop secretion-
inducing conditions (37°C, low Ca2?). Cells were lysed, and culture
supernatants were harvested, filtered, and precipitated with trichloro-
acetic acid. (a) The overall protein profile of the supernatant was
determined by Coomassie brilliant blue staining. Molecular masses in
kDa are indicated to the left. (b) Levels of YopE, YopH, YopJ, and
YopT were assessed by immunoblotting in cells and culture superna-
tants. Blots are representative of 3 independent experiments. The
relative density of each band compared to wild type is indicated. RpoA
in whole-cell lysates was used as a loading control. (c) qRT-PCR of
yopE, yopH, yopJ, and yopT transcripts. Bars represent the relative
average fold change of each transcript compared to wild type for 3
independent experiments. There were no significant differences be-
tween wild-type and mutant transcript levels for all yop genes tested.
2042SCHIANO ET AL.INFECT. IMMUN.
lular survival of a number of bacterial species. Indeed, the loss
of Hfq results in an increase in phagocytosis of Y. pestis, as well
as decreased intracellular survival (14). Conversely, there is no
effect on the intracellular survival of L. monocytogenes and F.
tularensis in the absence of Hfq (9, 32, 40, 42). Uropathogenic
E. coli does not have a defect in adherence or invasion of
cultured host cells, despite decreased colonization of the uri-
nary tract and bladder tissue in a mouse model of infection
(26). Given these results, it would be worthwhile to investigate
if there is a common pathway for intracellular survival that is
influenced by Hfq and its interactions with a particular set of
conserved sRNAs or if the mechanism is unique to each or-
Many species of pathogenic bacteria use the T3SS to inject
effector proteins into host cells, and type III secretion is es-
sential for the virulence of Yersinia species (10). Furthermore,
Yersinia species have been shown to target immune cells for
injection with type III effectors (25, 29); therefore, the defects
in persistence of the ?hfq mutant that we see in the Peyer’s
patches, lymph nodes, and spleen could be related to dysregu-
lation of the T3SS, as these organs are centers for lymphocytes.
Our data show a coordinated decrease in the production of
four Yop proteins (YopE, YopH, YopJ, and YopT) in the
absence of Hfq, which indicates that Hfq may play a role in the
regulation of Yop proteins directly or indirectly through inter-
actions with a regulator of T3S effectors. For example, the
protein LcrF coordinately regulates production of the effector
Yops at the transcriptional level in response to temperature
changes in an “all-or-none” fashion (20, 52). Furthermore,
Rosenzweig et al. have shown that in Yersinia spp., polynucle-
otide phosphorylase (PNPase) is a negative regulator of the
T3SS, while others have shown that the loss of Hfq in E. coli
leads to increased PNPase activity, which together may indi-
cate a role for Hfq on T3S via interactions with PNPase (34a,
It is possible that the decreased levels of T3S effectors in the
?hfq strain could account entirely for the inability of Y. pseudo-
tuberculosis to cause death of the mouse through decreased
fitness within the immune cell-containing lymphoid organs.
Mice infected with the T3S mutant (pYV?) displayed no
weight loss and showed no signs of illness in our intragastric
model of infection, whereas mice infected with the ?hfq strain
did lose weight and did show signs of illness, including hud-
dling and decreased activity (Fig. 2b and data not shown). This
suggests that Hfq may play a role in the regulation of other
virulence factors beyond the T3SS. Furthermore, given the
pleiotropic nature of Hfq, it is reasonable to anticipate that
there are other targets of Hfq and sRNAs involved in virulence
pathways. However, the slight production of Yop proteins seen
in the ?hfq strain by immunoblotting may be sufficient enough
to cause the level of illness seen in our mouse model (Fig. 10b);
therefore, we cannot rule out changes to T3S as the sole con-
tributor to the virulence defect.
We have demonstrated that the loss of Hfq significantly
attenuates Y. pseudotuberculosis in a mouse model of infection
and leads to defects in motility, intracellular survival, and type
III secretion. Further analysis of these phenotypes will reveal
the mechanisms by which Hfq and sRNAs mediate these ef-
fects in Y. pseudotuberculosis. Additionally, an analysis of the
host response to Y. pseudotuberculosis in the presence and
absence of Hfq may reveal if the disconnect between survival
and colonization is based on an Hfq-dependent, host-mediated
response. Finally, an understanding of the changes in protein
expression in the absence of Hfq will reveal the targets of
sRNAs that are regulated in an Hfq-dependent manner.
We thank James Bliska for his gift of the Yop antibodies and Mela-
nie Marketon for the RpoA antibody, Karla Satchell for helpful dis-
cussions and advice, Carl Waltenbaugh for photography, and Jovanka
Koo for help with animals.
This work was supported by the Searle Leadership Fund of North-
western University, the Northwestern University Feinberg School of
Medicine, and the NIH/NIAID Regional Center of Excellence for
Bio-defense and Emerging Infectious Diseases Research (RCE) Pro-
gram. We acknowledge membership within and support from the Re-
gion V “Great Lakes” RCE (NIH award 2-U54-AI-057153).
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Editor: J. B. Bliska
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