The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

In Vitro and In Vivo Evaluation of Lacticaseibacillus rhamnosus GG and Bifidobacterium lactis Bb12 Against Avian Pathogenic Escherichia coli and Identification of Novel Probiotic-Derived Bioactive Peptides.

Avian pathogenic E. coli (APEC), an extra-intestinal pathogenic E. coli (ExPEC), causes colibacillosis in poultry and is also a potential foodborne zoonotic pathogen. Currently, APEC infections in poultry are controlled by antibiotic medication; however, the emergence of multi-drug-resistant APEC strains and increased restrictions on the use of antibiotics in food-producing animals necessitate the development of new antibiotic alternative therapies. Here, we tested the anti-APEC activity of multiple commensal and probiotic bacteria in an agar-well diffusion assay and identified Lacticaseibacillus rhamnosus GG and Bifidobacterium lactis Bb12 producing strong zone of inhibition against APEC. In co-culture assay, L. rhamnosus GG and B. lactis Bb12 completely inhibited the APEC growth by 24 h. Further investigation revealed that antibacterial product(s) in the culture supernatants of L. rhamnosus GG and B. lactis Bb12 were responsible for the anti-APEC activity. The analysis of culture supernatants using LC-MS/MS identified multiple novel bioactive peptides (VQAAQAGDTKPIEV, AFDNTDTSLDSTFKSA, VTDTSGKAGTTKISNV, and AESSDTNLVNAKAA) in addition to the production of lactic acid. The oral administration (108 CFU/chicken) of L. rhamnosus GG significantly (P < 0.001) reduced the colonization (~ 1.6 logs) of APEC in the cecum of chickens. Cecal microbiota analysis revealed that L. rhamnosus GG moderated the APEC-induced alterations of the microbial community in the cecum of chickens. Further, L. rhamnosus GG decreased (P < 0.05) the abundance of phylum Proteobacteria, particularly those belonging to Enterobacteriaceae (Escherichia-Shigella) family. These studies indicate that L. rhamnosus GG is a promising probiotic to control APEC infections in chickens. Further studies are needed to optimize the delivery of L. rhamnosus GG in feed or water and in conditions simulating the field to facilitate its development for commercial applications.

Identification and characterization of novel small molecule inhibitors to control Mycoplasma gallisepticum infection in chickens.

Mycoplasma gallisepticum (MG) causes chronic respiratory disease in chickens, leading to severe economic losses to the poultry industry. Currently the disease is managed with antimicrobials and vaccination; however, emergence of multi-drug resistant Mycoplasma and the limited effect of vaccines necessitate development of novel approaches. A library of 4,182 small molecules (SMs) was screened for identification of narrow spectrum anti-MG compounds using high throughput screening. A total of 584 SMs were identified. Ten SMs possessed low MICs (0.78-100 µM) with efficacy against multiple MG strains and MG biofilm. These 10 SMs did not affect commensal/probiotic bacteria and other avian and foodborne pathogens. They displayed no or little toxicity on the avian macrophage HD-11 cells, human epithelial Caco-2 cells, and chicken red blood cells (RBCs); but, they were effective in reducing MG in chicken RBCs. Six SMs (SM1, SM3-5, and SM9-10) were tested in three-week-old chickens infected with MG (nasal spray; 109 CFU/bird). SM4 and SM9 reduced airsacculitis by 77.2 % and 82.9 %, MG load in the trachea by 0.9 log (p < 0.05) and 2.7 log (p < 0.0001), and tracheal mucosal thickness by 23 % and 61 %, respectively with no impact on the richness and evenness of the cecal (P = 0.6; H = 1.0) and tracheal (P = 0.8; H = 0.8) microbiota compared to the MG-infected controls. Both SM4 and SM9 treatments resulted in a significant alteration in the cell membrane conformation of MG. In conclusion; we identified two novel growth inhibitors of MG that are effective in chickens. These findings will facilitate development of novel antibacterials to control mycoplasmosis in poultry.

How the gut microbiome regulates host immune responses to viral vaccines.

The co-evolution of the microbiota and immune system has forged a mutually beneficial relationship. This relationship allows the host to maintain the balance between active immunity to pathogens and vaccines and tolerance to self-antigens and food antigens. In children living in low-income and middle-income countries, undernourishment and repetitive gastrointestinal infections are associated with the failure of oral vaccines. Intestinal dysbiosis associated with these environmental influences, as well as some host-related factors, compromises immune responses and negatively impacts vaccine efficacy. To understand how immune responses to viral vaccines can be optimally modulated, mechanistic studies of the relationship between the microbiome, host genetics, viral infections and the development and function of the immune system are needed. We discuss the potential role of the microbiome in modulating vaccine responses in the context of a growing understanding of the relationship between the gastrointestinal microbiota, host related factors (including histo-blood group antigens) and resident immune cell populations.

Transducer-Like Protein in Campylobacter jejuni With a Role in Mediating Chemotaxis to Iron and Phosphate.

Chemotaxis-mediated motility enables Campylobacter jejuni to navigate through complex environmental gradients and colonize diverse niches. C. jejuni is known to possess several methyl accepting chemotaxis proteins (MCPs), also called transducer-like proteins (Tlps). While the role of some of the Tlps in chemotaxis has been identified, their regulation and role in virulence is still not very clear. Here, we investigated the contribution of Tlp2 to C. jejuni chemotaxis, stress survival and colonization of the chicken gastrointestinal tract. The Δtlp2 deletion mutant showed decreased chemotaxis toward aspartate, pyruvate, inorganic phosphate (Pi), and iron (FeSO4). Transcriptional analysis of tlp2 with a promoter fusion reporter assay revealed that the tlp2 promoter (P tlp2 ) was induced by Pi and iron, both in the ferrous (Fe2+) and ferric form (Fe3+). RT-PCR analysis using overlapping primers indicated that the phoX gene, located immediately downstream of tlp2, is co-transcribed with tlp2. A transcription start site was identified at 53 bp upstream of the tlp2 start codon. The Δtlp2 mutant showed decreased colonization of the chicken gastrointestinal tract. Collectively, our findings revealed that the tlp2 plays a role in C. jejuni pathogenesis and colonization in the chicken host and its expression is regulated by iron.

E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro.

Microencapsulation enhances the oral delivery of probiotic bacteria. In this study, the probiotic Escherichia coli Nissle 1917 (EcN) was microencapsulated using alginate and chitosan nanoparticles. The result showed 90% encapsulation yield of EcN, and the encapsulated EcN displayed significantly (P < 0.05) increased survival in low pH (1.5), high bile salt concentration (4%), and high temperature (70 °C). The most effective cryopreservatives of EcN during freezing and thawing was skim milk and sucrose. Exposure to microencapsulated EcN significantly (P < 0.05) reduced the Campylobacter jejuni growth by 2 log CFU. The rate of EcN release from microcapsule was 9.2 × 105 cell min-1, and the appropriate model to describe its release kinetics was zero order. Importantly, the entrapment of EcN inside the microcapsule did not eliminate the exterior diffusion of EcN produced antioxidant compounds. In addition, the EcN microcapsule efficiently adhered to intestinal HT-29 cells and the pre-treatment of HT-29 cells with EcN-microcapsule for 4 h significantly (P < 0.05) reduced the invasion (1.9 log) of C. jejuni; whereas, completely abolished the intracellular survival. Furthermore, HT-29 cells pre-treated with encapsulated EcN in PCR array showed decreased expression (> 1.5-fold) of genes encoding chemokines, toll-like receptors, interleukins, and tumor necrosis factors. In conclusion, the alginate-chitosan microcapsule can provide effectual platform to deliver probiotic EcN and thereby can reduce the Campylobacter infection in chickens and humans.

Polyphosphate and associated enzymes as global regulators of stress response and virulence in Campylobacter jejuni.

Campylobacter jejuni (C. jejuni), a Gram-negative microaerophilic bacterium, is a predominant cause of bacterial foodborne gastroenteritis in humans worldwide. Despite its importance as a major foodborne pathogen, our understanding of the molecular mechanisms underlying C. jejuni stress survival and pathogenesis is limited. Inorganic polyphosphate (poly P) has been shown to play significant roles in bacterial resistance to stress and virulence in many pathogenic bacteria. C. jejuni contains the complete repertoire of enzymes required for poly P metabolism. Recent work in our laboratory and others have demonstrated that poly P controls a plethora of C. jejuni properties that impact its ability to survive in the environment as well as to colonize/infect mammalian hosts. This review article summarizes the current literature on the role of poly P in C. jejuni stress survival and virulence and discusses on how poly P-related enzymes can be exploited for therapeutic/prevention purposes. Additionally, the review article identifies potential areas for future investigation that would enhance our understanding of the role of poly P in C. jejuni and other bacteria, which ultimately would facilitate design of effective therapeutic/preventive strategies to reduce not only the burden of C. jejuni-caused foodborne infections but also of other bacterial infections in humans.

Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN-, LGG-, and EcN + LGG-colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK-cell function and decreased frequencies of apoptotic and TLR4+ mononuclear cells (MNCs). Consistent with the highest NK-cell activity, splenic CD172+ MNCs (DC enriched fraction) of EcN-colonized pigs produced the highest levels of IL-12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG-colonized pigs, suggesting that probiotics modulate each other's effects. Additionally, in vitro EcN-treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN-α, IL-12, and IL-10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN-mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC-IL-12-NK immune axis.

Polyphosphate kinases modulate Campylobacter jejuni outer membrane constituents and alter its capacity to invade and survive in intestinal epithelial cells in vitro.

Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection.

Transducer like proteins of Campylobacter jejuni 81-176: role in chemotaxis and colonization of the chicken gastrointestinal tract.

Transducer Like Proteins (Tlps), also known as methyl accepting chemotaxis proteins (MCP), enable enteric pathogens to respond to changing nutrient levels in the environment by mediating taxis toward or away from specific chemoeffector molecules. Despite recent advances in the characterization of chemotaxis responses in Campylobacter jejuni, the impact of Tlps on the adaptation of this pathogen to disparate niches and hosts is not fully characterized. The latter is particularly evident in the case of C. jejuni 81-176, a strain that is known to be highly invasive. Furthermore, the cytoplasmic group C Tlps (Tlp5, 6, and 8) were not extensively evaluated. Here, we investigated the role of C. jejuni 81-176 Tlps in chemotaxis toward various substrates, biofilm formation, in vitro interaction with human intestinal cells, and chicken colonization. We found that the Δtlp6 and Δtlp10 mutants exhibited decreased chemotaxis toward aspartate, whereas the Δtlp6 mutant displayed a decreased chemotaxis toward Tri-Carboxylic Acid (TCA) cycle intermediates such as pyruvate, isocitrate, and succinate. Our findings also corroborated that more than one Tlp is involved in mediating chemotaxis toward the same nutrient. The deletion of tlps affected important phenotypes such as motility, biofilm formation, and invasion of human intestinal epithelial cells (INT-407). The Δtlp8 mutant displayed increased motility in soft agar and showed decreased biofilm formation. The Δtlp8 and Δtlp9 mutants were significantly defective in invasion in INT-407 cells. The Δtlp10 mutant was defective in colonization of the chicken proximal and distal gastrointestinal tract, while the Δtlp6 and Δtlp8 mutants showed reduced colonization of the duodenum and jejunum. Our results highlight the importance of Tlps in C. jejuni's adaptation and pathobiology.

Polyphosphate-mediated modulation of Campylobacter jejuni biofilm growth and stability.

Biofilms increase C. jejuni's resilience to detergents, antibiotics, and environmental stressors. In these investigations, we studied the modulation of biofilm in response to phosphate related stressors. We found that the deletion of ppk1, phoX, and ppk2 (polyphosphate associated [poly P] genes) in C. jejuni modulated different stages of biofilm formation such as attached microcolonies, air-liquid biofilms, and biofilm shedding. Additionally, inorganic phosphate also modulated attached microcolonies, air-liquid biofilms, and biofilm shedding both independently of and additively in the poly P associated mutants. Furthermore, we observed that these different biofilm stages were affected by biofilm age: for example, the adherent microcolonies were maximum on day 2, while biofilm growth at the air-liquid interface and shedding was highest on day 3. Also, we observed altered calcofluor white reactive polysaccharides in poly P-associated mutants, as well as increased secretion of autoinducer-2 (AI-2) quorum sensing molecules in the ∆ppk2 mutant. Further, the polysaccharide and flagellar biosynthesis genes, that are associated with biofilm formation, were altered in these poly P-associated mutants. We conclude that the phosphate limiting condition modulates C. jejuni biofilm formation.

The impairment of methylmenaquinol:fumarate reductase affects hydrogen peroxide susceptibility and accumulation in Campylobacter jejuni.

The methylmenaquinol:fumarate reductase (Mfr) of Campylobacter jejuni is a periplasmic respiratory (redox) protein that contributes to the metabolism of fumarate and displays homology to succinate dehydrogenase (Sdh). Since chemically oxidized redox-enzymes, including fumarate reductase and Sdh, contribute to the generation of oxidative stress in Escherichia coli, we assessed the role of Mfr in C. jejuni after exposure to hydrogen peroxide (H2 O2 ). Our results show that a Mfr mutant (∆mfrA) strain was less susceptible to H2 O2 as compared to the wildtype (WT). Furthermore, the H2 O2 concentration in the ∆mfrA cultures was significantly higher than that of WT after exposure to the oxidant. In the presence of H2 O2 , catalase (KatA) activity and katA expression were significantly lower in the ∆mfrA strain as compared to the WT. Exposure to H2 O2 resulted in a significant decrease in total intracellular iron in the ∆mfrA strain as compared to WT, while the addition of iron to the growth medium mitigated H2 O2 susceptibility and accumulation in the mutant. The ∆mfrA strain was significantly more persistent in RAW macrophages as compared to the WT. Scanning electron microscopy showed that infection with the ∆mfrA strain caused prolonged changes to the macrophages' morphology, mainly resulting in spherical-shaped cells replete with budding structures and craters. Collectively, our results suggest a role for Mfr in maintaining iron homeostasis in H2 O2 stressed C. jejuni, probably via affecting the concentrations of intracellular iron.

Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni.

The inorganic polyphosphate (poly-P) is a key regulator of stress responses and virulence in many bacterial pathogens including Campylobacter jejuni. The role of exopolyphosphatases/guanosine pentaphosphate (pppGpp) phosphohydrolases (PPX/GPPA) in poly-P homeostasis and C. jejuni pathobiology remains unexplored. Here, we analyzed deletion mutants (∆ppx1, ∆ppx2) and the double knockout mutant (dkppx), all ∆ppx mutants exhibited increased capacity to accumulate poly-P; however only ∆ppx1 and dkppx mutants showed decreased accumulation of ppGpp, an alarmone molecule that regulates stringent response in bacteria, suggesting potential dual role for PPX1/GPPA. Nutrient survival defect of ∆ppx mutants was rescued by the supplementation of specific amino acids implying that survival defect may be associated with decreased ppGpp and/ or increased poly-P in ∆ppx mutants. The ppk1 and spoT were upregulated in both ∆ppx1 and ∆ppx2 suggesting a compensatory role for SpoT and Ppk1 in poly-P and ppGpp homeostasis. The lack of ppx genes resulted in defects in motility, biofilm formation, nutrient stress survival, invasion and intracellular survival indicating that maintaining a certain level of poly-P is critical for ppx genes in C. jejuni pathophysiology. Both ppx1 and ppx2 mutants were resistant to human complement-mediated killing; however, the dkppx mutant was sensitive. The serum susceptibility did not occur in the presence of MgCl 2 and EGTA suggesting an involvement of the classical or lectin pathway of complement mediated killing. Interestingly, the chicken serum did not have any effect on the ∆ppx mutants' survival. The observed serum susceptibility was not related to C. jejuni surface capsule and lipooligosaccharide structures. Our study underscores the importance of PPX/GPPA proteins in poly-P and ppGpp homeostasis, two critical molecules that modulate environmental stress responses and virulence in C. jejuni.

Lactobacilli and bifidobacteria promote immune homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs.

The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.

Of energy and survival incognito: a relationship between viable but non-culturable cells formation and inorganic polyphosphate and formate metabolism in Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative food-borne bacterium that can cause mild to serious diseases in humans. A variety of stress conditions including exposure to formic acid, a weak organic acid, can cause C. jejuni to form viable but non-culturable cells (VBNC), which was proposed as a potential survival mechanism. The inability to detect C. jejuni VBNC using standard culturing techniques may increase the risk of exposure to foods contaminated with this pathogen. However, little is known about the cellular mechanisms and triggers governing VBNC formation. Here, we discuss novel mechanisms that potentially affect VBNC formation in C. jejuni and emphasize the impact of formic acid on this process. Specifically, we highlight findings that show that impairing inorganic polyphosphate (poly-P) metabolism reduces the ability of C. jejuni to form VBNC in a medium containing formic acid. We also discuss the potential effect of poly-P and formate metabolism on energy homeostasis and cognate VBNC formation. The relationship between poly-P metabolism and VBNC formation under acid stress has only recently been identified and may represent a breakthrough in understanding this phenomenon and its impact on food safety.

Erythritol triggers expression of virulence traits in Brucella melitensis.

Erythritol is a four-carbon sugar preferentially utilized by Brucella spp. The presence of erythritol in the placentas of goats, cows, and pigs has been used to explain the localization of Brucella to these sites and the subsequent accumulation of large amounts of bacteria, eventually leading to abortion. Here we show that Brucella melitensis will also localize to an artificial site of erythritol within a mouse, providing a potential model system to study the pathogenesis of Brucella abortion. Immunohistological staining of the sites of erythritol within infected mice indicated a higher than expected proportion of extracellular bacteria. Ensuing experiments suggested intracellular B. melitensis was unable to replicate within macrophages in the presence of erythritol and that erythritol was able to reach the site of intracellular bacteria. The intracellular inhibition of growth was found to encourage the bacteria to replicate extracellularly rather than intracellularly, a particularly interesting development in Brucella pathogenesis. To determine the effect of erythritol on expression of B. melitensis genes, bacteria grown either with or without erythritol were analyzed by microarray. Two major virulence pathways were up-regulated in response to exposure to erythritol (the type IV secretion system VirB and flagellar proteins), suggesting a role for erythritol in virulence.

Respiratory proteins contribute differentially to Campylobacter jejuni's survival and in vitro interaction with hosts' intestinal cells.

BACKGROUND: The genetic features that facilitate Campylobacter jejuni's adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni's adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni's i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens). RESULTS: C. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA's motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H(2)O(2)), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H(2)O(2) was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division. CONCLUSIONS: We conclude that the RPs contribute to C. jejuni's motility, H(2)O(2) resistance, biofilm formation, and in vitro interactions with hosts' intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen's remarkable potential for adaptation.

Contribution of TAT system translocated PhoX to Campylobacter jejuni phosphate metabolism and resilience to environmental stresses.

Campylobacter jejuni is a common gastrointestinal pathogen that colonizes food animals; it is transmitted via fecal contamination of food, and infections in immune-compromised people are more likely to result in serious long-term illness. Environmental phosphate is likely an important sensor of environmental fitness and the ability to obtain extracellular phosphate is central to the bacteria's core metabolic responses. PhoX is the sole alkaline phosphatase in C. jejuni, a substrate of the TAT transport system. Alkaline phosphatases mediate the hydrolytic removal of inorganic phosphate (Pi) from phospho-organic compounds and thereby contribute significantly to the polyphosphate kinase 1 (ppk1) mediated formation of poly P, a molecule that regulates bacterial response to stresses and virulence. Similarly, deletion of the tatC gene, a key component of the TAT system, results in diverse phenotypes in C. jejuni including reduced stress tolerance and in vivo colonization. Therefore, here we investigated the contribution of phoX in poly P synthesis and in TAT-system mediated responses. The phoX deletion mutant showed significant decrease (P<0.05) in poly P accumulation in stationary phase compared to the wild-type, suggesting that PhoX is a major contributor to the inorganic phosphate pool in the cell which is essential for poly P synthesis. The phoX deletion is sufficient for a nutrient stress defect similar to the defect previously described for the ΔtatC mutant. Additionally, the phoX deletion mutant has increased resistance to certain antimicrobials. The ΔphoX mutant was also moderately defective in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization. Further, the ΔphoX mutant produced increased biofilm that can be rescued with 1 mM inorganic phosphate. The qRT-PCR of the ΔphoX mutant revealed transcriptional changes that suggest potential mechanisms for the increased biofilm phenotype.