Proteomic Identification of saeRS-Dependent Targets Critical for Protective Humoral Immunity against Staphylococcus aureus Skin Infection

Recurrent Staphylococcus aureus skin and soft tissue infections (SSTIs) are common despite detectable antibody responses, leading to the belief that the immune response elicited by these infections is not protective. We recently reported that S. aureus USA300 SSTI elicits antibodies that protect against recurrent SSTI in BALB/c but not C57BL/6 mice, and in this study, we aimed to uncover the specificity of the protective antibodies. Using a proteomic approach, we found that S. aureus SSTI elicited broad polyclonal antibody responses in both BALB/c and C57BL/6 mice and identified 10 S. aureus antigens against which antibody levels were significantly higher in immune BALB/c serum. Four of the 10 antigens identified are regulated by the saeRS operon, suggesting a dominant role for saeRS in protection. Indeed, infection with USA300Δsae failed to protect against secondary SSTI with USA300, despite eliciting a strong polyclonal antibody response against antigens whose expression is not regulated by saeRS. Moreover, the antibody repertoire after infection with USA300Δsae lacked antibodies specific for 10 saeRS-regulated antigens, suggesting that all or a subset of these antigens are necessary to elicit protective immunity. Infection with USA300Δhla elicited modest protection against secondary SSTI, and complementation of USA300Δsae with hla restored protection but incompletely. Together, these findings support a role for both Hla and other saeRS-regulated antigens in eliciting protection and suggest that host differences in immune responses to saeRS-regulated antigens may determine whether S. aureus infection elicits protective or nonprotective immunity against recurrent infection.     

Staphylococcus aureus Serotype 5 Capsular Polysaccharide Is Antiphagocytic and Enhances Bacterial Virulence in a Murine Bacteremia Model

Controversy persists over the role that the capsular polysaccharide plays in the pathogenesis of Staphylococcus aureus infections. To address this issue, we compared the mouse virulence of S. aureus Reynolds and capsule-defective mutant strains cultivated under conditions of high or low capsule expression. Strain Reynolds cells cultivated on Columbia salt agar plates expressed ~100-fold more type 5 capsular polysaccharide than did cells cultivated in Columbia salt broth. The relative virulence of strain Reynolds and its capsule-defective mutants after growth on either solid or liquid medium was examined in mice challenged intraperitoneally or intravenously. The results indicated that agar-grown Reynolds cells were cleared from the bloodstream of mice less readily than broth-grown Reynolds cells. When the parental and mutant strains were cultivated on solid medium, strain Reynolds sustained a higher level of bacteremia than did the capsular mutants. We performed in vitro opsonophagocytic killing assays to determine whether staphylococcal virulence for mice correlated with resistance to phagocytosis. S. aureus Reynolds cultivated on solid medium was susceptible to phagocytic killing only in the presence of specific capsular antibodies and complement. Strain Reynolds grown in broth showed opsonic requirements for phagocytic killing that were similar to those of the capsular mutants (grown in broth or on agar); i.e., the bacteria were opsonized for phagocytosis by nonimmune serum with complement activity. These studies indicate that optimal expression of capsule enhances bacterial virulence in the mouse model of bacteremia, probably by rendering the organisms resistant to opsonophagocytic killing by leukocytes.     

Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages

Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1Δ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1Δ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1Δ mutant is due to its inability to leave the lung. The fbp1Δ mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.     

Staphylococcus aureus Hyaluronidase Is a CodY-Regulated Virulence Factor

Staphylococcus aureus is a Gram-positive pathogen that causes a diverse range of bacterial infections. Invasive S. aureus strains secrete an extensive arsenal of hemolysins, immunomodulators, and exoenzymes to cause disease. Our studies have focused on the secreted enzyme hyaluronidase (HysA), which cleaves the hyaluronic acid polymer at the β-1,4 glycosidic bond. In the study described in this report, we have investigated the regulation and contribution of this enzyme to S. aureus pathogenesis. Using the Nebraska Transposon Mutant Library (NTML), we identified eight insertions that modulate extracellular levels of HysA activity. Insertions in the sigB operon, as well as in genes encoding the global regulators SarA and CodY, significantly increased HysA protein levels and activity. By altering the availability of branched-chain amino acids, we further demonstrated CodY-dependent repression of HysA activity. Additionally, through mutation of the CodY binding box upstream of hysA, the repression of HysA production was lost, suggesting that CodY is a direct repressor of hysA expression. To determine whether HysA is a virulence factor, a ΔhysA mutant of a community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 strain was constructed and found to be attenuated in a neutropenic, murine model of pulmonary infection. Mice infected with this mutant strain exhibited a 4-log-unit reduction in bacterial burden in their lungs, as well as reduced lung pathology and increased levels of pulmonary hyaluronic acid, compared to mice infected with the wild-type, parent strain. Taken together, these results indicate that S. aureus hyaluronidase is a CodY-regulated virulence factor.     

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.     

Evaluation of Protective Efficacy of Live Attenuated Salmonella enterica Serovar Gallinarum Vaccine Strains against Fowl Typhoid in Chickens

Salmonella enterica serovar Gallinarum is the etiological agent of fowl typhoid, which constitutes a considerable economic problem for poultry growers in developing countries. The vaccination of chickens seems to be the most effective strategy to control the disease in those areas. We constructed S. Gallinarum strains with a deletion of the global regulatory gene fur and evaluated their virulence and protective efficacy in Rhode Island Red chicks and Brown Leghorn layers. The fur deletion mutant was avirulent and, when delivered orally to chicks, elicited excellent protection against lethal S. Gallinarum challenge. It was not as effective when given orally to older birds, although it was highly immunogenic when delivered by intramuscular injection. We also examined the effect of a pmi mutant and a combination of fur deletions with mutations in the pmi and rfaH genes, which affect O-antigen synthesis, and ansB, whose product inhibits host T-cell responses. The S. Gallinarum Δpmi mutant was only partially attenuated, and the ΔansB mutant was fully virulent. The Δfur Δpmi and Δfur ΔansB double mutants were attenuated but not protective when delivered orally to the chicks. However, a Δpmi Δfur strain was highly immunogenic when administered intramuscularly. All together, our results show that the fur gene is essential for the virulence of S. Gallinarum, and the fur mutant is effective as a live recombinant vaccine against fowl typhoid.     

Site-Specific Mutation of the Sensor Kinase GraS in Staphylococcus aureus Alters the Adaptive Response to Distinct Cationic Antimicrobial Peptides

The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ΔgraS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.     

Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.     

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a ΔsrtA Δbps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Δbps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the ΔsrtA Δbps and Δbps mutants were ~2 log₁₀ greater than that of OG1RF or the ΔsrtA mutant. Similarly, ~2 log₁₀ fewer bacteria were recovered from the kidneys after infection with the Δbps mutant (P = 0.017) and the ΔsrtA Δbps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Δbps mutant was slightly, but not significantly, less attenuated than the ΔsrtA Δbps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).     

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.     

The Small RNA RyhB Contributes to Siderophore Production and Virulence of Uropathogenic Escherichia coli

In Escherichia coli, the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenic E. coli strain, their impact on the production of virulence-associated factors is still unknown for a pathogenic E. coli strain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenic E. coli (UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion of fur alone did not attenuate virulence, whereas a ΔryhB mutant and a Δfur ΔryhB double mutant showed significantly reduced bladder colonization. The Δfur mutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the ΔryhB mutant produced lower levels of siderophores. This decrease was correlated with the downregulation of shiA (encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) and iucD (involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine. iucD was also downregulated in bladders infected with the ΔryhB mutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenic E. coli.     

Identification and Functional Characterization of the Novel Edwardsiella tarda Effector EseJ

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) of E. tarda has been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ΔesaN mutant, we identified a new effector by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 of E. tarda strain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ΔeseJ mutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ΔeseJ mutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ΔeseJ mutant elicits higher production of reactive oxygen species than wild-type E. tarda. The replication defect is consistent with the attenuation of the ΔeseJ mutant in the blue gourami fish model: the 50% lethal dose (LD₅₀) of the ΔeseJ mutant is 2.34 times greater than that of the wild type, and the ΔeseJ mutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.     

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.     

Staphopains Modulate Staphylococcus aureus Biofilm Integrity

Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ΔsigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture.     

Evaluation of Brilliance MRSA 2 Agar for Detection of Methicillin-Resistant Staphylococcus aureus in Clinical Samples

We compared 2 chromogenic media (Oxoid Brilliance MRSA 2 agar [Thermo Fisher Scientific] and MRSA-ID [bioMérieux]) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in 1,368 hospital samples. For both media, broth enrichment was essential to obtain satisfactory diagnostic performance. Although with direct cultures only, the diagnostic performance (particularly sensitivity) of Brilliance MRSA 2 agar appears better than that of MRSA-ID, no difference in sensitivity or specificity between the media was detected after inclusion of an enrichment step.