Plasmid-Encoded MCP Is Involved in Virulence,Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage.     

The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host

Bacterial pathogens produce a number of autotransporters that possess diverse functions. These include the family of serine protease autotransporters of Enterobacteriaceae (SPATEs) produced by enteric pathogens such as Shigella flexneri and enteroaggregative Escherichia coli. Of these SPATEs, one termed “protein involved in colonization,” or Pic, has been shown to possess mucinase activity in vitro, but to date, its role in in vivo enteric pathogenesis is unknown. Testing a pic null (ΔpicC) mutant in Citrobacter rodentium, a natural mouse pathogen, found that the C. rodentium ΔpicC strain was impaired in its ability to degrade mucin in vitro compared to the wild type. Upon infection of mice, the ΔpicC mutant exhibited a hypervirulent phenotype with dramatically heavier pathogen burdens found in intestinal crypts. ΔpicC mutant-infected mice suffered greater barrier disruption and more severe colitis and weight loss, necessitating their euthanization between 10 and 14 days postinfection. Notably, the virulence of the ΔpicC mutant was normalized when the picC gene was restored; however, a PicC point mutant causing loss of mucinase activity did not replicate the ΔpicC phenotype. Exploring other aspects of PicC function, the ΔpicC mutant was found to aggregate to higher levels in vivo than wild-type C. rodentium. Moreover, unlike the wild type, the C. rodentium ΔpicC mutant had a red, dry, and rough (RDAR) morphology in vitro and showed increased activation of the innate receptor Toll-like receptor 2 (TLR2). Interestingly, the C. rodentium ΔpicC mutant caused a degree of pathology similar to that of wild-type C. rodentium when infecting TLR2-deficient mice, showing that despite its mucinase activity, PicC''s major role in vivo may be to limit C. rodentium''s stimulation of the host''s innate immune system.     

Interplay between the QseC and QseE Bacterial Adrenergic Sensor Kinases in Salmonella enterica Serovar Typhimurium Pathogenesis

The bacterial adrenergic sensor kinases QseC and QseE respond to epinephrine and/or norepinephrine to initiate a complex phosphorelay regulatory cascade that modulates virulence gene expression in several pathogens. We have previously shown that QseC activates virulence gene expression in Salmonella enterica serovar Typhimurium. Here we report the role of QseE in S. Typhimurium pathogenesis as well as the interplay between these two histidine sensor kinases in gene regulation. An S. Typhimurium qseE mutant is hampered in the invasion of epithelial cells and intramacrophage replication. The ΔqseC strain is highly attenuated for intramacrophage survival but has only a minor defect in invasion. However, the ΔqseEC strain has only a slight attenuation in invasion, mirroring the ΔqseC strain, and has an intermediary intramacrophage replication defect in comparison to the ΔqseE and ΔqseC strains. The expressions of the sipA and sopB genes, involved in the invasion of epithelial cells, are activated by epinephrine via QseE. The expression levels of these genes are still decreased in the ΔqseEC double mutant, albeit to a lesser extent, congruent with the invasion phenotype of this mutant. The expression level of the sifA gene, important for intramacrophage replication, is decreased in the qseE mutant and the ΔqseEC double mutant grown in vitro. However, as previously reported by us, the epinephrine-dependent activation of this gene occurs via QseC. In the systemic model of S. Typhimurium infection of BALB/c mice, the qseC and qseE mutants are highly attenuated, while the double mutant has an intermediary phenotype. Altogether, these data suggest that both adrenergic sensors play an important role in modulating several aspects of S. Typhimurium pathogenesis.     

ERK Regulates Renal Cell Proliferation and Renal Cyst Expansion in inv Mutant Mice

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invΔC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invΔC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants.     

An Inducible and Secreted Eukaryote-Like Serine/Threonine Kinase of Salmonella enterica Serovar Typhi Promotes Intracellular Survival and Pathogenesis

Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs in Salmonella enterica serovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of the t4519 locus of Salmonella Typhi Ty2 have all the signatures of eukaryotic superfamily kinases. t4519 encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activities in vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis of Salmonella Typhi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519 strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK of Salmonella Typhi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in the in vivo pathogenesis of Salmonella spp.     

The Borrelia hermsii Factor H Binding Protein FhbA Is Not Required for Infectivity in Mice or for Resistance to Human Complement In Vitro

The primary causative agent of tick-borne relapsing fever in North America is Borrelia hermsii. It has been hypothesized that B. hermsii evades complement-mediated destruction by binding factor H (FH), a host-derived negative regulator of complement. In vitro, B. hermsii produces a single FH binding protein designated FhbA (FH binding protein A). The properties and ligand binding activity of FhbA suggest that it plays multiple roles in pathogenesis. It binds plasminogen and has been identified as a significant target of a B1b B cell-mediated IgM response in mice. FhbA has also been explored as a potential diagnostic antigen for B. hermsii infection in humans. The ability to test the hypothesis that FhbA is a critical virulence factor in vivo has been hampered by the lack of well-developed systems for the genetic manipulation of the relapsing fever spirochetes. In this report, we have successfully generated a B. hermsii fhbA deletion mutant (the B. hermsii YORΔfhbA strain) through allelic exchange mutagenesis. Deletion of fhbA abolished FH binding by the YORΔfhbA strain and eliminated cleavage of C3b on the cell surface. However, the YORΔfhbA strain remained infectious in mice and retained resistance to killing in vitro by human complement. Collectively, these results indicate that B. hermsii employs an FhbA/FH-independent mechanism of complement evasion that allows for resistance to killing by human complement and persistence in mice.     

Role of flagellin and the two-component CheA/CheY system of Listeria monocytogenes in host cell invasion and virulence

The flagellum protein flagellin of Listeria monocytogenes is encoded by the flaA gene. Immediately downstream of flaA, two genes, cheY and cheA, encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in flaA. cheY, and cheA to elucidate their role in the biology of infection with L. monocytogenes. The ΔcheY, ΔcheA, and double-mutant ΔcheYA mutants, but not ΔflaA mutant, were motile in liquid media. However, the ΔcheA mutant had impaired swarming and the ΔcheY and ΔcheYA mutants were unable to swarm on soft agar plates, suggesting that cheY and cheA genes encode proteins involved in chemotaxis. The ΔflaA, ΔcheY, ΔcheA, and ΔcheYA mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the ΔflaA and ΔcheYA mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the ΔflaA or ΔcheYA mutant. In summary, we show that listerial flaA. cheY, and cheA gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that flaA could also be involved in the triggering of immune responses.     

Titan Cell Production Enhances the Virulence of Cryptococcus neoformans

Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4Δ gpr5Δ mutant, which has minimal titan cell production in vivo. The gpr4Δ gpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4Δ gpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.     

Investigation of the Function of Candida albicans Als3 by Heterologous Expression in Candida glabrata

During hematogenously disseminated infection, blood-borne Candida albicans invades the endothelial cell lining of the vasculature to invade the deep tissues. Although the C. albicans Als3 invasin is critical for invasion and damage of endothelial cells in vitro, a C. albicans als3Δ/Δ mutant has normal virulence in the mouse model of disseminated infection. We hypothesized that the contribution of Als3 to virulence is obscured by the presence of additional C. albicans invasins. To elucidate the in vivo function of Als3, we heterologously expressed C. albicans ALS3 in Candida glabrata, a yeast that lacks a close ALS3 ortholog and has low virulence in mice. We found that following intravenous inoculation into mice, the ALS3-expressing strain preferentially trafficked to the brain, where it induced significantly elevated levels of myeloperoxidase, tumor necrosis factor, monocyte chemoattractant protein 1, and gamma interferon. Also, the ALS3-expressing strain had enhanced adherence to and invasion of human brain microvascular endothelial cells in vitro, demonstrating a potential mechanism for ALS3-mediated neurotropism. In addition, upon initiation of infection, the ALS3-expressing strain had increased trafficking to the cortex of the kidneys. With prolonged infection, this strain persisted in the kidneys at significantly higher levels than the control strain but did not induce an elevated inflammatory response. Finally, the ALS3-expressing strain had increased resistance to neutrophil killing in vitro. These results indicate that during disseminated infection, Als3 mediates initial trafficking to the brain and renal cortex and contributes to fungal persistence in the kidneys.     

Phosphatidylcholine-Specific Phospholipase C from Listeria monocytogenes Is an Important Virulence Factor in Murine Cerebral Listeriosis

Meningoencephalitis is a serious and often fatal complication of Listeria monocytogenes infection. The aim of the present study was to analyze the role of internalin A (InlA) and B, which are involved in the invasion of L. monocytogenes into cultivated host tissue cells, and that of phosphatidylcholine-specific phospholipase C (PlcB), which mainly promotes the direct cell-to-cell spread of L. monocytogenes, in murine cerebral listeriosis by use of an InlA/B (ΔinlAB2)- and a PlcB (ΔplcB2)-deficient isogenic deletion mutant strain and the wild-type (WT) L. monocytogenes EGD. Listeria strains were directly applied to the brain, a technique which has been employed previously to study the pathogenesis of cerebral listeriosis (D. Schlüter, S. B. Oprisiu, S. Chahoud, D. Weiner, O. D. Wiestler, H. Hof, and M. Deckert-Schlüter, Eur. J. Immunol. 25:2384–2391, 1995). We demonstrated that PlcB, but not InlA or InlB, is an important virulence factor in cerebral listeriosis. Nonimmunized mice infected intracerebrally with the ΔplcB2 strain survived significantly longer and had a reduced intracerebral bacterial load compared to mice infected with the ΔinlAB2 strain or WT bacteria. In addition, immunization with the WT prior to intracerebral infection significantly increased the survival rate of mice challenged intracerebrally with the ΔplcB2 strain compared to that of mice infected with the WT or ΔinlAB2 strain. Histopathology revealed that the major difference between the various experimental groups was a significantly delayed intracerebral spread of the ΔplcB2 mutant strain, indicating that cell-to-cell spread is an important pathogenic feature of cerebral listeriosis. Interestingly, irrespective of the Listeria mutant used, the apoptosis of hippocampal and cerebellar neurons and an internal hydrocephalus developed in surviving mice, indicating that these complications are not dependent on the virulence factors InlA/B and PlcB. In conclusion, this study points to PlcB as a virulence factor important for the intracerebral pathogenesis of murine L. monocytogenes meningoencephalitis.     

Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.     

Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium

Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes of Enterobacteriaceae family members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp in Yersinia pestis virulence in mouse models of bubonic and pneumonic plague, we characterized an msbB in-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a Δlpp ΔmsbB double mutant of the highly virulent Y. pestis CO92 strain. Although the ΔmsbB single mutant was minimally attenuated, the Δlpp single mutant and the Δlpp ΔmsbB double mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the Δlpp ΔmsbB double mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the Δlpp ΔmsbB double mutant, but not the Δlpp or ΔmsbB single mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the Δlpp ΔmsbB double mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of the msbB gene augmented the attenuation of the Δlpp mutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the Δlpp ΔmsbB double mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future.     

PI3K-dependent host cell actin rearrangements are required for Cronobacter sakazakii invasion of human brain microvascular endothelial cells

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that can cause neonatal sepsis and meningitis. The mechanism involved in the pathogenesis of C. sakazakii meningitis remains largely unknown. Previous studies indicated that bacterial invasion of brain microvascular endothelial cells is required for penetration into the central nervous system. In this study, we found that C. sakazakii invasion of human brain microvascular endothelial cells (HBMEC) was significantly inhibited by cytochalasin D, a disrupting agent of actin microfilaments. Disassembly of actin stress fibers and cortical actin fibers was observed in HBMEC infected with C. sakazakii. C. sakazakii infection leads to increased Akt phosphorylation in HBMEC, which was blocked by treatment with PI3K inhibitors. Meanwhile, PI3K and Akt inhibitors significantly inhibited C. sakazakii invasion of HBMEC. Our further results illustrated that the C. sakazakii-induced Akt activation and C. sakazakii invasion were attenuated in HBMEC transfected with dominant-negative PI3K (Δp110). More importantly, the actin filaments rearrangements in HBMEC induced by C. sakazakii were effectively blocked by PI3K inhibitors treatment and transfection with Δp110. Taken together, our findings demonstrated that PI3K-mediated actin rearrangements are required for C. sakazakii invasion of HBMEC.     

The Mycobacterium avium subsp. paratuberculosis MAP3464 gene encodes an oxidoreductase involved in invasion of bovine epithelial cells through the activation of host cell Cdc42

Mycobacterium avium subsp. paratuberculosis infection of cattle takes place through the intestinal mucosa. To identify M. avium subsp. paratuberculosis genes associated with the invasion of bovine epithelial cells in vitro, we screened a library of transposon mutants. Several mutants of M. avium subsp. paratuberculosis were identified which invaded Madin-Darby bovine kidney (MDBK) epithelial cells less efficiently than wild-type (wt) M. avium subsp. paratuberculosis. The ΔOx mutant had the transposon located in the MAP3464 gene, a putative oxidoreductase gene whose expression is upregulated upon bacterial contact with MDBK cells. Complete restoration of invasion comparable to that for the wt bacterium was achieved by introducing a copy of the complete oxidoreductase operon into the ΔOx mutant. Immunoprecipitation and Western blot analysis indicated that wt M. avium subsp. paratuberculosis activates Cdc42 and RhoA pathways of internalization 15 and 60 min after infection of the host cell, respectively. The ΔOx mutant, however, failed to activate the Cdc42 pathway. To determine whether an M. avium subsp. paratuberculosis protein delivered to the host cell mediates the entry of the wt bacterium by activation of the Cdc42 pathway, affinity precipitation of active Cdc42 from MDBK-infected cells followed by mass spectrometry was carried out. We identified a 17-amino-acid bacterial peptide associated with the Cdc42 of cells infected with wt M. avium subsp. paratuberculosis but not with the ΔOx mutant. The sequence of the peptide matches MAP3985c, a hypothetical protein, possibly functioning as a putative Cdc42 effector. These findings reveal a novel signaling pathway activated during M. avium subsp. paratuberculosis entry that links the product of MAP3464 gene to activation of Cdc42 in the host cell.     

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD₅₀) equivalent to 6,800 LD₅₀s of WT CO92. The mutant-infected animals developed balanced T_H1- and T_H2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD₅₀s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant''s sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.