Synoviolin is not a pathogenic factor for auto-inflammatory diseases.

Auto-inflammatory syndromes are rare diseases characterized by arthritis and joint destruction, symptoms similar to but distinct from rheumatoid arthritis (RA). Therapeutic targets have not been well characterized for auto-inflammatory syndromes, although the E3 ligase Synoviolin was previously shown to be a novel therapeutic target for RA. Here, we show that Synoviolin loss has little impact on a model of auto-inflammatory diseases. We previously established such a model, the hIL-1 cTg mouse, in which IL-1 signaling was constitutively activated, and animals exhibit symptoms recapitulating auto-inflammatory syndromes such as major joint dominant arthritis. Here, we crossed hIL-1 cTg with Synoviolin flox'd mice to yield hIL-1 cTg/Synoviolin cKO mice. Synoviolin gene expression was ablated in adult hIL-1 cTg/Synoviolin cKO mice by injection of pIpC to activate Mx1 promoter-driven Cre recombinase. However, symptoms seen in hIL-1 cTg mice such as arthritis and joint destruction were not alleviated by targeting Synoviolin, ruling out Synoviolin as a therapeutic target for auto-inflammatory disease. Our results indicate that although similar, RA and auto-inflammatory diseases are different diseases, and treatment strategies should differ accordingly.

Porphyromonas gingivalis enhances pneumococcal adhesion to human alveolar epithelial cells by increasing expression of host platelet-activating factor receptor.

Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet-activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR-specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp-dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this.

Secreted protein MoHrip2 is required for full virulence of Magnaporthe oryzae and modulation of rice immunity.

MoHrip2, identified from Magnaporthe oryzae as an elicitor, can activate plant defense responses either in the form of recombinant protein in vitro or ectopic expressed protein in rice. However, its intrinsic function in the infective interaction of M. oryzae-rice is largely unknown. Here, we found that mohrip2 expression was significantly induced at stages of fungal penetration and colonization. Meanwhile, the induced MoHrip2 mainly accumulated in the rice apoplast by outlining the entire invasive hyphae during infection, and its secretion was via the conventional endoplasmic reticulum (ER)-to-Golgi pathway, demonstrating the nature of MoHrip2 as an apoplastic effector. What's more, the disease facilitating function of MoHrip2 was revealed by the significantly compromised virulence of Δmohrip2 mutants on rice seedlings and even on the wounded rice leaves. Inoculations of these mutant strains on rice leaf sheaths showed a reduction in penetration and subsequent expansion of fungal growth, which is probably due to activated host immunity including the expression of certain defense-related genes and the production of certain phytoalexins. Altogether, these results demonstrated the necessity of MoHrip2 in suppression of host immunity and the full virulence of M. oryzae.

The AI-2/luxS Quorum Sensing System Affects the Growth Characteristics, Biofilm Formation, and Virulence of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis) is a kind of opportunistic pathogen of the upper respiratory tract of piglets. Under certain circumstances, virulent strains can breach the mucosal barrier and enter the bloodstream, causing severe Glässer's disease. Many virulence factors are found to be related to the pathogenicity of H. parasuis strain, but the pathogenic mechanism remains unclear. LuxS/AI-2, as a kind of very important quorum sensing system, affects the growth characteristics, biofilm formation, antibiotic production, virulence, and metabolism of different strains. In order to investigate the effect of luxS/AI-2 quorum sensing system on the virulence of H. parasuis, a deletion mutant strain (ΔluxS) and complemented strain (C-luxS) were constructed and characterized. The results showed that the luxS gene participated in regulating and controlling stress resistance, biofilm formation and virulence. Compared with wild-type strain, ΔluxS strain decreased the production of AI-2 molecules and the tolerance toward oxidative stress and heat shock, and it reduced the abilities of autoagglutination, hemagglutination, and adherence, whereas it increased the abilities to form biofilm in vitro. In vivo experiments showed that ΔluxS strain attenuated its virulence about 10-folds and significantly decreased its tissue burden of bacteria in mice, compared with the wild-type strain. Taken together, the luxS/AI-2 quorum sensing system in H. parasuis not only plays an important role in growth and biofilm formation, but also affects the pathogenicity of H. parasuis.

Salmonella Outer Protein B Suppresses Colitis Development via Protecting Cell From Necroptosis.

Salmonella effectors translocated into epithelial cells contribute to the pathogenesis of infection. They mediate epithelial cell invasion and subsequent intracellular replication. However, their functions in vivo have not been well-identified. In this study, we uncovered a role for Salmonella outer protein B (SopB) in modulating necroptosis to facilitate bacteria escape epithelial cell and spread to systemic sites through a Salmonella-induced colitis model. Mice infected with SopB deleted strain ΔsopB displayed increased severity to colitis, reduced mucin expression and increased bacterial translocation. In vitro study, we found there was an increased goblet cell necroptosis following ΔsopB infection. Consistently, mice infected with ΔsopB had a strong upregulation of mixed lineage kinase domain-like (MLKL) phosphorylation. Deletion of MLKL rescued severity of tissue inflammatory, improved mucin2 expression and abolished the increased bacterial translocation in mice infected with ΔsopB. Intriguingly, the expression of sopB in LS174T cells was downregulated. The temporally regulated SopB expression potentially switched the role from epithelial cell invasion to bacterial transmission. Collectively, these results indicated a role for SopB in modulating the onset of necroptosis to increased bacteria pathogenesis and translocated to systemic sites.

The trimeric autotransporter adhesin BadA is required for in vitro biofilm formation by Bartonella henselae.

Bartonella henselae (Bh) is a Gram-negative rod transmitted to humans by a scratch from the common house cat. Infection of humans with Bh can result in a range of clinical diseases including lymphadenopathy observed in cat-scratch disease and more serious disease from persistent bacteremia. It is a common cause of blood-culture negative endocarditis as the bacterium is capable of growing as aggregates, and forming biofilms on infected native and prosthetic heart valves. The aggregative growth requires a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA). TAAs are found in all Bartonella species and many other Gram-negative bacteria. Using Bh Houston-1, Bh Houston-1 ∆badA and Bh Houston-1 ∆badA/pNS2PTrc badA (a partial complement of badA coding for a truncated protein of 741 amino acid residues), we analyze the role of BadA in adhesion and biofilm formation. We also investigate the role of environmental factors such as temperature on badA expression and biofilm formation. Real-time cell adhesion monitoring and electron microscopy show that Bh Houston-1 adheres and forms biofilm more efficiently than the Bh Houston-1 ∆badA. Deletion of the badA gene significantly decreases adhesion, the first step in biofilm formation in vitro, which is partially restored in Bh Houston-1 ∆badA/pNS2PTrc badA. The biofilm formed by Bh Houston-1 includes polysaccharides, proteins, and DNA components and is susceptible to enzymatic degradation of these components. Furthermore, both pH and temperature influence both badA expression and biofilm formation. We conclude that BadA is required for optimal adhesion, agglutination and biofilm formation.

Blocking ß-1,6-glucan synthesis by deleting KRE6 and SKN1 attenuates the virulence of Candida albicans.

ß-1,6-glucan is an important component of the fungal cell wall. The ß-1,6-glucan synthase gene KRE6 was thought to be essential in the fungal pathogen Candida albicans because it could not be deleted in previous efforts. Also, the role of its homolog SKN1 was unclear because its deletion caused no defects. Here, we report the construction and characterization of kre6Δ/Δ, skn1Δ/Δ and kre6Δ/Δ skn1Δ/Δ mutants in C. albicans. While deleting KRE6 or SKN1 had no obvious phenotypic consequence, deleting both caused slow growth, cell separation failure, cell wall abnormalities, diminished hyphal growth, poor biofilm formation and loss of virulence in mice. Furthermore, the GPI-linked cell surface proteins Hwp1 and the invasin Als3 or Ssa1 were not detected in kre6Δ/Δ skn1Δ/Δ mutant. In GMM medium, RT-qPCR and western blotting revealed a constitutive expression of KRE6 and growth conditions-associated activation of SKN1. Like many hypha-specific genes, SKN1 is repressed by Nrg1, but its activation does not involve the transcription factor Efg1. Dysregulation of SKN1 reduces C. albicans ability to damage epithelial and endothelial cells and attenuates its virulence. Given the vital role of ß-1,6-glucan synthesis in C. albicans physiology and virulence, Kre6 and Skn1 are worthy targets for developing antifungal agents.

The Meningococcal Cysteine Transport System Plays a Crucial Role in Neisseria meningitidis Survival in Human Brain Microvascular Endothelial Cells.

While Neisseria meningitidis typically exists in an asymptomatic nasopharyngeal carriage state, it may cause potentially lethal diseases in humans, such as septicemia or meningitis, by invading deeper sites in the body. Since the nutrient compositions of human cells are not always conducive to meningococci, N. meningitidis needs to exploit nutrients from host environments. In the present study, the utilization of cysteine by the meningococcal cysteine transport system (CTS) was analyzed for the pathogenesis of meningococcal infections. A N. meningitidis strain deficient in one of the three cts genes annotated as encoding cysteine-binding protein (cbp) exhibited approximately 100-fold less internalization into human brain microvascular endothelial cells (HBMEC) than the wild-type strain. This deficiency was restored by complementation with the three cts genes together, and the infectious phenotype of HBMEC internalization correlated with cysteine uptake activity. However, efficient accumulation of ezrin was observed beneath the cbp mutant. The intracellular survival of the cbp mutant in HBMEC was markedly reduced, whereas equivalent reductions of glutathione concentrations and of resistance to reactive oxygens species in the cbp mutant were not found. The cbp mutant grew well in complete medium but not in synthetic medium supplemented with less than 300 µM cysteine. Taking cysteine concentrations in human cells and other body fluids, including blood and cerebrospinal fluid, into consideration, the present results collectively suggest that the meningococcal CTS is crucial for the acquisition of cysteine from human cells and participates in meningococcal nutrient virulence.IMPORTANCENeisseria meningitidis colonizes at a nasopharynx of human as a unique host and has many strains that are auxotrophs for amino acids for their growth. To cause invasive meningococcal diseases (IMD) such as sepsis and meningitis, N. meningitidis passes through epithelial and endothelial barriers and infiltrates into blood and cerebrospinal fluid as well as epithelial and endothelial cells. However, meningococcal nutrients, including cysteine, become less abundant when it more deeply infiltrates the human body even during inflammation, such that N. meningitidis has to acquire nutrients in order to survive/persist, disseminate, and proliferate in humans. This was the first study to examine the relationship between meningococcal cysteine acquisition and the pathogenesis of meningococcal infections. The results of the present study provide insights into the mechanisms by which pathogens with auxotrophs acquire nutrients in hosts and may also contribute to the development of treatments and prevention strategies for IMD.

CitE Enzymes Are Essential for Mycobacterium tuberculosis to Establish Infection in Macrophages and Guinea Pigs.

Bacterial citrate lyase activity has been demonstrated in various eukaryotes, bacteria and archaea, underscoring their importance in energy metabolism of the cell. While the bacterial citrate lyase comprises of three different subunits, M. tuberculosis genome lacks CitD and CitF subunits of citrate lyase complex but encodes for 2 homologs of CitE subunits, Rv2498c and Rv3075c. Using temperature sensitive mycobacteriophages, we were able to generate both single and double citE mutant strains of M. tuberculosis. The survival experiments revealed increased susceptibility of the double mutant strain to oxidative stress in comparison to the parental strain. Also, simultaneous deletion of both citE1 and citE2 in M. tuberculosis genome resulted in impairment of intracellular replication in macrophages. The double mutant strain displayed reduced growth in lungs and spleens of guinea pigs. This is the first study demonstrating that M. tuberculosis critically requires CitE subunits of citrate lyase for pathogenesis. Taken together, these findings position these enzymes as potential targets for development of anti-tubercular small molecules.

The Involvement of the Cas9 Gene in Virulence of Campylobacter jejuni.

Campylobacter jejuni is considered as the leading cause of gastroenteritis all over the world. This bacterium has the CRISPR-cas9 system, which is used as a gene editing technique in different organisms. However, its role in bacterial virulence has just been discovered; that discovery, however, is just the tip of the iceberg. The purpose of this study is to find out the relationship between cas9 and virulence both phenotypically and genotypically in C. jejuni NCTC11168. Understanding both aspects of this relationship allows for a much deeper understanding of the mechanism of bacterial pathogenesis. The present study determined virulence in wild and mutant strains by observing biofilm formation, motility, adhesion and invasion, intracellular survivability, and cytotoxin production, followed by the transcriptomic analysis of both strains. The comparative gene expression profile of wild and mutant strains was determined on the basis of De-Seq transcriptomic analysis, which showed that the cas9 gene is involved in enhancing virulence. Differential gene expression analysis revealed that multiple pathways were involved in virulence, regulated by the CRISPR-cas9 system. Our findings help in understanding the potential role of cas9 in regulating the other virulence associated genes in C. jejuni NCTC11168. The findings of this study provide critical information about cas9's potential involvement in enhancing the virulence of C. jejuni, which is a major public health threat.

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis.

Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.

Salmonella Typhimurium Invalidated for the Three Currently Known Invasion Factors Keeps Its Ability to Invade Several Cell Models.

To establish an infection, Salmonella has to interact with eukaryotic cells. Invasion of non-phagocytic cells (i.e., epithelial, fibroblast and endothelial cells) involves either a trigger or a zipper mechanism mediated by the T3SS-1 or the invasin Rck, respectively. Another outer membrane protein, PagN, was also implicated in the invasion. However, other unknown invasion factors have been previously suggested. Our goal was to evaluate the invasion capability of a Salmonella Typhimurium strain invalidated for the three known invasion factors. Non-phagocytic cell lines of several animal origins were tested in a gentamicin protection assay. In most cells, we observed a drastic decrease in the invasion rate between the wild-type and the triple mutant. However, in five cell lines, the triple mutant invaded cells at a similarly high level to the wild-type, suggesting the existence of unidentified invasion factors. For the wild-type and the triple mutant, scanning-electron microscopy, confocal imaging and use of biochemical inhibitors confirmed their cellular uptake and showed a zipper-like mechanism of internalization involving both clathrin- and non-clathrin-dependent pathways. Despite a functional T3SS-1, the wild-type bacteria seemed to use the same entry route as the mutant in our cell model. All together, these results demonstrate the existence of unknown Salmonella invasion factors, which require further characterization.

Role of class II P fimbriae and cytokine response in the pathogenesis of Escherichia coli kidney infection in diabetic mice.

BACKGROUND: The role of class II P fimbriae (P fimbriae II) in diabetic kidney infections is uncertain, although some genetic and epidemiological studies suggest a lower prevalence of P fimbriae II genes in Escherichia coli strains isolated from diabetic patients with complicated kidney infections. METHODS: We inoculated a P fimbriae II deficient E. coli (DH5αT) or an isogenic P fimbriae II expressing transformant (DH5αTP) into the bladders of diabetic and non-diabetic BALB/C mice, and sacrificed them after 3 days. The incidence of bladder or kidney infection (≥103 CFU of E. coli per bladder or kidney), bacteremia (≥102 CFU of E. coli on blood culture plate), kidney pathological score, immunoreactive Histo-score (H-score), and corrected H-score (H-score adjusted for Log10 CFU of bacteria in the kidney) were compared among groups. RESULTS: Diabetic mice were more susceptible to bladder infection than non-diabetic mice with both transformants. The geometric mean of bacteria counts in kidneys was significantly increased only when the diabetic mice were infected with DH5αTP. Among the 4 groups of mice, diabetic mice infected with DH5αTP had the highest incidence of kidney infection and bacteremia, and the highest renal pathology scores. The IL-8 H-score and the corrected IL-6 and IL-8 H-score were significantly lower in diabetic than non-diabetic mice. CONCLUSION: We concluded that P fimbriae II contribute to the pathogenesis and severity of E. coli kidney infections in diabetic mice. An impaired cytokine response may also contribute to the increased incidence and severity of kidney infections in diabetic hosts.

General response of Salmonella enterica serovar Typhimurium to desiccation: A new role for the virulence factors sopD and sseD in survival.

Salmonella can survive for long periods under extreme desiccation conditions. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular regulation of this adaptation mechanism. To determine the genetic components involved in Salmonella's cellular response to desiccation, we performed a global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low water activity (aw 0.11) and cells equilibrated to high water activity (aw 1.0). The analysis revealed that 719 genes were differentially regulated between the two conditions, of which 290 genes were up-regulated at aw 0.11. Most of these genes were involved in metabolic pathways, transporter regulation, DNA replication/repair, transcription and translation, and, more importantly, virulence genes. Among these, we decided to focus on the role of sopD and sseD. Deletion mutants were created and their ability to survive desiccation and exposure to aw 0.11 was compared to the wild-type strain and to an E. coli O157:H7 strain. The sopD and sseD mutants exhibited significant cell viability reductions of 2.5 and 1.3 Log (CFU/g), respectively, compared to the wild-type after desiccation for 4 days on glass beads. Additional viability differences of the mutants were observed after exposure to aw 0.11 for 7 days. E. coli O157:H7 lost viability similarly to the mutants. Scanning electron microscopy showed that both mutants displayed a different morphology compared to the wild-type and differences in production of the extracellular matrix under the same conditions. These findings suggested that sopD and sseD are required for Salmonella's survival during desiccation.

Mutation of the cyclic di-GMP phosphodiesterase gene in Burkholderia lata SK875 attenuates virulence and enhances biofilm formation.

Burkholderia sp. is a gram-negative bacterium that commonly exists in the environment, and can cause diseases in plants, animals, and humans. Here, a transposon mutant library of a Burkholderia lata isolate from a pig with swine respiratory disease in Korea was screened for strains showing attenuated virulence in Caenorhabditis elegans. One such mutant was obtained, and the Tn5 insertion junction was mapped to rpfR, a gene encoding a cyclic di-GMP phosphodiesterase that functions as a receptor. Mutation of rpfR caused a reduction in growth on CPG agar and swimming motility as well as a rough colony morphology on Congo red agar. TLC analysis showed reduced AHL secretion, which was in agreement with the results from plate-based and bioluminescence assays. The mutant strain produced significantly more biofilm detected by crystal violet staining than the parent strain. SEM of the mutant strain clearly showed that the overproduced biofilm contained a filamentous structure. These results suggest that the cyclic di-GMP phosphodiesterase RpfR plays an important role in quorum sensing modulation of the bacterial virulence and biofilm formation.

A Nonredundant Phosphopantetheinyl Transferase, PptA, Is a Novel Antifungal Target That Directs Secondary Metabolite, Siderophore, and Lysine Biosynthesis in Aspergillus fumigatus and Is Critical for Pathogenicity.

Secondary metabolites are key mediators of virulence for many pathogens. Aspergillus fumigatus produces a vast array of these bioactive molecules, the biosynthesis of which is catalyzed by nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs). Both NRPSs and PKSs harbor carrier domains that are primed for acceptance of secondary metabolic building blocks by a phosphopantetheinyl transferase (P-pant). The A. fumigatus P-pant PptA has been shown to prime the putative NRPS Pes1 in vitro and has an independent role in lysine biosynthesis; however, its role in global secondary metabolism and its impact on virulence has not been described. Here, we demonstrate that PptA has a nonredundant role in the generation of the vast majority of detectable secondary metabolites in A. fumigatus, including the immunomodulator gliotoxin, the siderophores triacetylfusarinine C (TAFC) and ferricrocin (FC), and dihydroxy naphthalene (DHN)-melanin. We show that both the lysine and iron requirements of a pptA null strain exceed those freely available in mammalian tissues and that loss of PptA renders A. fumigatus avirulent in both insect and murine infection models. Since PptA lacks similarity to its mammalian orthologue, we assert that the combined role of this enzyme in both primary and secondary metabolism, encompassing multiple virulence determinants makes it a very promising antifungal drug target candidate. We further exemplify this point with a high-throughput fluorescence polarization assay that we developed to identify chemical inhibitors of PptA function that have antifungal activity.IMPORTANCE Fungal diseases are estimated to kill between 1.5 and 2 million people each year, which exceeds the global mortality estimates for either tuberculosis or malaria. Only four classes of antifungal agents are available to treat invasive fungal infections, and all suffer pharmacological shortcomings, including toxicity, drug-drug interactions, and poor bioavailability. There is an urgent need to develop a new class of drugs that operate via a novel mechanism of action. We have identified a potential drug target, PptA, in the fungal pathogen Aspergillus fumigatus PptA is required to synthesize the immunotoxic compound gliotoxin, DHN-melanin, which A. fumigatus employs to evade detection by host cells, the amino acid lysine, and the siderophores TAFC and FC, which A. fumigatus uses to scavenge iron. We show that strains lacking the PptA enzyme are unable to establish an infection, and we present a method which we use to identify novel antifungal drugs that inactivate PptA.

Capsular polysaccharide production and serum survival of Vibrio vulnificus are dependent on antitermination control by RfaH.

The human pathogen Vibrio vulnificus undergoes phase variation among colonial morphotypes, including a virulent opaque form which produces capsular polysaccharide (CPS) and a translucent phenotype that produces little or no CPS and is attenuated. Here, we found that a V. vulnificus mutant defective for RfaH antitermination control showed a diminished capacity to undergo phase variation and displayed significantly reduced distal gene expression within the Group I CPS operon. Moreover, the rfaH mutant produced negligible CPS and was highly sensitive to killing by normal human serum, results which indicate that RfaH is likely essential for virulence in this bacterium.

Protective Immunity Elicited by Oral Immunization of Mice with Salmonella enterica Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants.

We evaluated the extent of attenuation and immunogenicity of the ΔlppAB and ΔlppAB ΔmsbB mutants of Salmonella enterica serovar Typhimurium when delivered to mice by the oral route. These mutants were deleted either for the Braun lipoprotein genes (lppA and lppB) or in combination with the msbB gene, which encodes an acetyltransferase required for lipid A modification of lipopolysaccharide. Both the mutants were attenuated (100% animal survival) and triggered robust innate and adaptive immune responses. Comparable levels of IgG and its isotypes were produced in mice infected with wild-type (WT) S. typhimurium or its aforementioned mutant strains. The ΔlppAB ΔmsbB mutant-immunized animals resulted in the production of higher levels of fecal IgA and serum cytokines during later stages of vaccination (adaptive response). A significant production of interleukin-6 from T-cells was also noted in the ΔlppAB ΔmsbB mutant-immunized mice when compared to that of the ΔlppAB mutant. On the other hand, IL-17A production was significantly more in the serum of ΔlppAB mutant-immunized mice (innate response) with a stronger splenic T-cell proliferative and tumor-necrosis factor-α production. Based on 2-dimensional gel analysis, alterations in the levels of several proteins were observed in both the mutant strains when compared to that in WT S. typhimurium and could be associated with the higher immunogenicity of the mutants. Finally, both ΔlppAB and ΔlppAB ΔmsbB mutants provided complete protection to immunized mice against a lethal oral challenge dose of WT S. typhimurium. Thus, these mutants may serve as excellent vaccine candidates and also provide a platform for delivering heterologous antigens.

Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.

Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate disease.

Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus.