Effects of Limosilactobacillus reuteri strains PTA-126787 and PTA-126788 on intestinal barrier integrity and immune homeostasis in an alcohol-induced leaky gut model.

Intestinal barrier is a first line of defense that prevents entry of various harmful substances from the lumen into the systemic environment. Impaired barrier function with consequent translocation of harmful substances into systemic circulation ("leaky gut") is a central theme in many gastrointestinal, autoimmune, mental, and metabolic diseases. Probiotics have emerged as a promising strategy to maintain intestinal integrity and address "leaky gut". Using in silico, in vitro and avian in vivo analyses, we previously showed that two novel L. reuteri strains, PTA-126787 (L. reuteri 3630) and PTA-126788 (L. reuteri 3632), isolated from broiler chickens possess favorable safety profiles. Consistent with a recent study, here we show that L. reuteri 3630 and 3632 are phylogenetically similar to human L. reuteri strains. Daily administration of high doses of L. reuteri 3630 and 3632 to Sprague Dawley rats for 28 days was found to be safe with no adverse effects. More importantly, administration of L. reuteri 3630 and 3632 significantly reduced markers associated with alcohol-induced leaky gut, by downregulating inflammatory cytokines and upregulating anti-inflammatory cytokines in an alcohol model of leaky gut in mice. While L. reuteri 3630 cells and supernatant showed no activation, L. reuteri 3632 cells but not supernatant showed activation of AhR, a key transcription factor that regulates gut and immune homeostasis. L. reuteri 3630 is creamish white in morphology typical of Lactobacillus species and L. reuteri 3632 displays a unique orange pigmentation, which was stable even after passaging for 480 generations. We identified a rare polyketide biosynthetic gene cluster in L. reuteri 3632 that likely encodes for the orange-pigmented secondary metabolite. Similar to L. reuteri 3632 cells, the purified orange metabolite activated AhR. All together, these data provide evidence on the phylogenetic relatedness, safety, efficacy, and one of the likely mechanisms of action of L. reuteri 3630 and 3632 for potential probiotic applications to address "leaky gut" and associated pathologies in humans.

In silico, in vitro and in vivo characterization of host-associated Latilactobacillus curvatus strains for potential probiotic applications in farmed Atlantic salmon (Salmo salar).

Salmon aquaculture is the fastest growing animal protein production system in the world; however, intensive farming leads to poor weight gain, stress, and disease outbreaks. Probiotics offer the potential to enhance growth performance and feed efficiency in Atlantic salmon, as well as immunostimulate fish against common pathogens, benefitting farmers and consumers with more efficient production. Here, we isolated and identified 900 native microbial isolates including 18 Lactobacilli from the farmed salmon intestines. Based on whole-genome sequencing and phylogenetic analysis, the Lactobacillus candidates belonged to Latilactobacillus curvatus (L. curvatus) species and formed two distinct phylogenetic groups. Using bioinformatics and in vitro analyses, we selected two candidates L. curvatus ATCC PTA-127116 and L. curvatus ATCC PTA-127117, which showed desirable safety and probiotic properties. The two L. curvatus candidates were evaluated for safety and efficacy (higher final weight) in Atlantic salmon alongside spore-forming Bacilli isolated from salmon, poultry, and swine. All the tested candidates were safe to salmon with no adverse effects. While we did not see efficacy in any Bacillus supplemented groups, compared to untreated group, the group administered with the two L. curvatus strains consortium in feed for seven weeks in freshwater showed indicators of improvement in final body weight by 4.2%. Similarly, the two L. curvatus candidates were also evaluated for safety and efficacy in Atlantic salmon in saltwater; the group administered with the two L. curvatus strains consortium in feed for 11 weeks showed indicators of improvement in final body weight by 4.7%. Comprehensive metabolomics analyses in the presence of different prebiotics and/or additives identified galactooligosaccharide as a potential prebiotic to enhance the efficacy of two L. curvatus candidates. All together, these data provide comprehensive genomic, phenotypic and metabolomic evidence of safety and desirable probiotic properties as well as indicators of in vivo efficacy of two novel endogenous L. curvatus candidates for potential probiotic applications in Atlantic salmon. The in vivo findings need to be confirmed in larger performance studies, including field trials.

Transcriptome Analyses of Chicken Primary Macrophages Infected With Attenuated Salmonella Typhimurium Mutants.

Salmonella enterica is one of the most common foodborne illnesses in the United States and worldwide, with nearly one-third of the cases attributed to contaminated eggs and poultry products. Vaccination has proven to be an effective strategy to reduce Salmonella load in poultry. The Salmonella Typhimurium Δcrp-cya (MeganVac1) strain is the most commonly used vaccine in the United States; however, the mechanisms of virulence attenuation and host response to this vaccine strain are poorly understood. Here, we profiled the invasion and intracellular survival phenotypes of Δcrp-cya and its derivatives (lacking key genes required for intra-macrophage survival) in HD11 macrophages and the transcriptome response in primary chicken macrophages using RNA-seq. Compared to the parent strain UK1, all the mutant strains were highly defective in metabolizing carbon sources related to the TCA cycle and had greater doubling times in macrophage-simulating conditions. Compared to UK1, the majority of the mutants were attenuated for invasion and intra-macrophage survival. Compared to Δcrp-cya, while derivatives lacking phoPQ, ompR-envZ, feoABC and sifA were highly attenuated for invasion and intracellular survival within macrophages, derivatives lacking ssrAB, SPI13, SPI2, mgtRBC, sitABCD, sopF, sseJ and sspH2 showed increased ability to invade and survive within macrophages. Transcriptome analyses of macrophages infected with UK1, Δcrp-cya and its derivatives lacking phoPQ, sifA and sopF demonstrated that, compared to uninfected macrophages, 138, 148, 153, 155 and 142 genes were differentially expressed in these strains, respectively. Similar changes in gene expression were observed in macrophages infected with these strains; the upregulated genes belonged to innate immune response and host defense and the downregulated genes belonged to various metabolic pathways. Together, these data provide novel insights on the relative phenotypes and early response of macrophages to the vaccine strain and its derivatives. The Δcrp-cya derivatives could facilitate development of next-generation vaccines with improved safety.

Growth promotion and antibiotic induced metabolic shifts in the chicken gut microbiome.

Antimicrobial growth promoters (AGP) have played a decisive role in animal agriculture for over half a century. Despite mounting concerns about antimicrobial resistance and demand for antibiotic alternatives, a thorough understanding of how these compounds drive performance is missing. Here we investigate the functional footprint of microbial communities in the cecum of chickens fed four distinct AGP. We find relatively few taxa, metabolic or antimicrobial resistance genes similarly altered across treatments, with those changes often driven by the abundances of core microbiome members. Constraints-based modeling of 25 core bacterial genera associated increased performance with fewer metabolite demands for microbial growth, pointing to altered nitrogen utilization as a potential mechanism of narasin, the AGP with the largest performance increase in our study. Untargeted metabolomics of narasin treated birds aligned with model predictions, suggesting that the core cecum microbiome might be targeted for enhanced performance via its contribution to host-microbiota metabolic crosstalk.

Recombinant Limosilactobacillus (Lactobacillus) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis.

Necrotic enteritis (NE), caused by Clostridium perfringens, is an intestinal disease with devastating economic losses to the poultry industry. NE is a complex disease and predisposing factors that compromise gut integrity are required to facilitate C. perfringens proliferation and toxin production. NE is also characterized by drastic shifts in gut microbiota; C. perfringens is negatively correlated with Lactobacilli. Vaccines are only partially effective against NE and antibiotics suffer from the concern of resistance development. These strategies address only some aspects of NE pathogenesis. Thus, there is an urgent need for alternative strategies that address multiple aspects of NE biology. Here, we developed Limosilactobacillus (Lactobacillus) reuteri vectors for in situ delivery of nanobodies against NetB and α toxin, two key toxins associated with NE pathophysiology. We generated nanobodies and showed that these nanobodies neutralize NetB and α toxin. We selected L. reuteri vector strains with intrinsic benefits and demonstrated that these strains inhibit C. perfringens and secrete over 130 metabolites, some of which play a key role in maintaining gut health. Recombinant L. reuteri strains efficiently secreted nanobodies and these nanobodies neutralized NetB. The recombinant strains were genetically and phenotypically stable over 480 generations and showed persistent colonization in chickens. A two-dose in ovo and drinking water administration of recombinant L. reuteri strains protected chickens from NE-associated mortality. These results provide proof-of-concept data for using L. reuteri as a live vector for delivery of nanobodies with broad applicability to other targets and highlight the potential synergistic effects of vector strains and nanobodies for addressing complex diseases such as NE.

In silico, in vitro and in vivo safety evaluation of Limosilactobacillus reuteri strains ATCC PTA-126787 & ATCC PTA-126788 for potential probiotic applications.

The last two decades have witnessed a tremendous growth in probiotics and in the numbers of publications on their potential health benefits. Owing to their distinguishing beneficial effects and long history of safe use, species belonging to the Lactobacillus genus are among the most widely used probiotic species in human food and dietary supplements and are finding increased use in animal feed. Here, we isolated, identified, and evaluated the safety of two novel Limosilactobacillus reuteri (L. reuteri) isolates, ATCC PTA-126787 & ATCC PTA-126788. More specifically, we sequenced the genomes of these two L. reuteri strains using the PacBio sequencing platform. Using a combination of biochemical and genetic methods, we identified the two strains as belonging to L. reuteri species. Detailed in silico analyses showed that the two strains do not encode for any known genetic sequences of concern for human or animal health. In vitro assays confirmed that the strains are susceptible to clinically relevant antibiotics and do not produce potentially harmful by-products such as biogenic amines. In vitro bile and acid tolerance studies demonstrated that the two strains have similar survival profiles as the commercial L. reuteri probiotic strain DSM 17938. Most importantly, daily administration of the two probiotic strains to broiler chickens in drinking water for 26 days did not induce any adverse effect, clinical disease, or histopathological lesions, supporting the safety of the strains in an in vivo avian model. All together, these data provide in silico, in vitro and in vivo evidence of the safety of the two novel candidates for potential probiotic applications in humans as well as animals.

Multi-Omics Characterization of Host-Derived Bacillus spp. Probiotics for Improved Growth Performance in Poultry.

Microbial feed ingredients or probiotics have been used widely in the poultry industry to improve production efficiency. Spore-forming Bacillus spp. offer advantages over traditional probiotic strains as Bacillus spores are resilient to high temperature, acidic pH, and desiccation. This results in increased strain viability during manufacturing and feed-pelleting processes, extended product shelf-life, and increased stability within the animal's gastrointestinal tract. Despite numerous reports on the use of Bacillus spores as feed additives, detailed characterizations of Bacillus probiotic strains are typically not published. Insufficient characterizations can lead to misidentification of probiotic strains in product labels, and the potential application of strains carrying virulence factors, toxins, antibiotic resistance, or toxic metabolites. Hence, it is critical to characterize in detail the genomic and phenotypic properties of these strains to screen out undesirable properties and to tie individual traits to clinical outcomes and possible mechanisms. Here, we report a screening workflow and comprehensive multi-omics characterization of Bacillus spp. for use in broiler chickens. Host-derived Bacillus strains were isolated and screened for desirable probiotic properties. The phenotypic, genomic and metabolomic analyses of three probiotic candidates, two Bacillus amyloliquefaciens (Ba ATCC PTA126784 and ATCC PTA126785), and a Bacillus subtilis (Bs ATCC PTA126786), showed that all three strains had promising probiotic traits and safety profiles. Inclusion of Ba ATCC PTA12684 (Ba-PTA84) in the feed of broiler chickens resulted in improved growth performance, as shown by a significantly improved feed conversion ratio (3.3%), increased of European Broiler Index (6.2%), and increased average daily gain (ADG) (3.5%). Comparison of the cecal microbiomes from Ba PTA84-treated and control animals suggested minimal differences in microbiome structure, indicating that the observed growth promotion presumably was not mediated by modulation of cecal microbiome.

Multiple Class I and Class II Haemophilus ducreyi Strains Cause Cutaneous Ulcers in Children on an Endemic Island.

Background: Together with Treponema pallidum subspecies pertenue, Haemophilus ducreyi is a major cause of exudative cutaneous ulcers (CUs) in children. For H. ducreyi, both class I and class II strains, asymptomatic colonization, and environmental reservoirs have been found in endemic regions, but the epidemiology of this infection is unknown. Methods: Based on published whole-genome sequences of H. ducreyi CU strains, a single-locus typing system was developed and applied to H. ducreyi-positive CU samples obtained prior to, 1 year after, and 2 years after the initiation of a mass drug administration campaign to eradicate CU on Lihir Island in Papua New Guinea. DNA from the CU samples was amplified with class I and class II dsrA-specific primers and sequenced; the samples were classified into dsrA types, which were geospatially mapped. Selection pressure analysis was performed on the dsrA sequences. Results: Thirty-seven samples contained class I sequences, 27 contained class II sequences, and 13 contained both. There were 5 class I and 4 class II types circulating on the island; 3 types accounted for approximately 87% of the strains. The composition and geospatial distribution of the types varied little over time and there was no evidence of selection pressure. Conclusions: Multiple strains of H. ducreyi cause CU on an endemic island and coinfections are common. In contrast to recent findings with T. pallidum pertenue, strain composition is not affected by antibiotic pressure, consistent with environmental reservoirs of H. ducreyi. Such reservoirs must be addressed to achieve eradication of H. ducreyi.

Evaluation of CpxRA as a Therapeutic Target for Uropathogenic Escherichia coli Infections.

CpxRA is an envelope stress response system found in all members of the family Enterobacteriaceae; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR in Escherichia coli by inhibiting CpxA phosphatase activity. As a prelude to testing such compounds in vivo, here we constructed cpxA (in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) and cpxR (system absent) deletion mutants of uropathogenic E. coli (UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between the cpxR mutant and its parent, but in the cpxA mutant, several UPEC virulence determinants were downregulated, including the fim and pap operons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cells in vitro In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented in trans Unexpectedly, in single-strain challenges, only the cpxA mutant was attenuated for virulence in the kidney but not in the bladder or urine. For the cpxA mutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.

Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection.

During infection, Neisseria gonorrhoeae senses and responds to stress; such responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system that is a homolog of CpxRA. In Escherichia coli, CpxRA senses and responds to envelope stress; CpxA is a sensor kinase/phosphatase for CpxR, a response regulator. When a cpxA mutant is grown in medium containing glucose, CpxR is phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J. L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszynski, R. W. Carlson, and W. M. Shafer, Antimicrob Agents Chemother 60:4690-4700, 2016, https://doi.org/10.1128/AAC.00823-16) showed that MisR (CpxR) is required for the maintenance of membrane integrity and resistance to antimicrobial peptides, suggesting a role in gonococcal survival in vivo Here, we evaluated the contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model of cervicovaginal colonization and identified MisR-regulated genes using RNA sequencing (RNA-Seq). The deletion of misR or misS severely reduced the capacity of N. gonorrhoeae to colonize mice or maintain infection over a 7-day period and reduced microbial fitness after exposure to heat shock. Compared to the wild type (WT), the inactivation of misR identified 157 differentially regulated genes, most of which encoded putative envelope proteins. The inactivation of misS identified 17 differentially regulated genes compared to the WT and 139 differentially regulated genes compared to the misR mutant, 111 of which overlapped those differentially expressed in the comparison of the WT versus the misR mutant. These data indicate that an intact MisRS system is required for gonococcal infection of mice. Provided the MisR is constitutively phosphorylated in the misS mutant, the data suggest that controlled but not constitutive activation is required for gonococcal infection in mice.

Neisseria meningitidis ST11 Complex Isolates Associated with Nongonococcal Urethritis, Indiana, USA, 2015-2016.

At a clinic in Indianapolis, Indiana, USA, we observed an increase in Neisseria gonorrhoeae-negative men with suspected gonococcal urethritis who had urethral cultures positive for N. meningitidis. We describe genomes of 2 of these N. meningitidis sequence type 11 complex urethritis isolates. Clinical evidence suggests these isolates may represent an emerging urethrotropic clade.

Haemophilus ducreyi Cutaneous Ulcer Strains Diverged from Both Class I and Class II Genital Ulcer Strains: Implications for Epidemiological Studies.

BACKGROUND: Haemophilus ducreyi has emerged as a major cause of cutaneous ulcers (CU) in yaws-endemic regions of the tropics in the South Pacific, South East Asia and Africa. H. ducreyi was once thought only to cause the genital ulcer (GU) disease chancroid; GU strains belong to 2 distinct classes, class I and class II. Using whole-genome sequencing of 4 CU strains from Samoa, 1 from Vanuatu and 1 from Papua New Guinea, we showed that CU strains diverged from the class I strain 35000HP and that one CU strain expressed ß-lactamase. Recently, the Center for Disease Control and Prevention released the genomes of 11 additional CU strains from Vanuatu and Ghana; however, the evolutionary relationship of these CU strains to previously-characterized CU and GU strains is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We performed phylogenetic analysis of 17 CU and 10 GU strains. Class I and class II GU strains formed two distinct clades. The class I strains formed two subclades, one containing 35000HP and HD183 and the other containing the remainder of the class I strains. Twelve of the CU strains formed a subclone under the class I 35000HP subclade, while 2 CU strains formed a subclone under the other class I subclade. Unexpectedly, 3 of the CU strains formed a subclone under the class II clade. Phylogenetic analysis of dsrA-hgbA-ncaA sequences yielded a tree similar to that of whole-genome phylogenetic tree. CONCLUSIONS/SIGNIFICANCE: CU strains diverged from multiple lineages within both class I and class II GU strains. Multilocus sequence typing of dsrA-hgbA-ncaA could be reliably used for epidemiological investigation of CU and GU strains. As class II strains grow relatively poorly and are relatively more susceptible to vancomycin than class I strains, these findings have implications for methods to recover CU strains. Comparison of contemporary CU and GU isolates would help clarify the relationship between these entities.

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.

Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers.

Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. MunsonJr, E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans.

Draft Whole-Genome Sequence of Haemophilus ducreyi Strain AUSPNG1, Isolated from a Cutaneous Ulcer of a Child from Papua New Guinea.

Haemophilus ducreyi has recently emerged as a leading cause of cutaneous ulcers in the yaws-endemic areas of Papua New Guinea and other South Pacific islands. Here, we report the draft genome sequence of the H. ducreyi strain AUSPNG1, isolated from a cutaneous ulcer of a child from Papua New Guinea.

Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains.

BACKGROUND: Although cutaneous ulcers (CU) in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU) and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin? METHODOLOGY/PRINCIPAL FINDINGS: To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya) and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin. CONCLUSIONS/SIGNIFICANCE: These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions.

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.

Haemophilus ducreyi RpoE and CpxRA appear to play distinct yet complementary roles in regulation of envelope-related functions.

Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi, does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNA-Seq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi.

Correcting imbalanced reads coverage in bacterial transcriptome sequencing with extreme deep coverage.

High throughput bacterial RNA-Seq experiments can generate extremely high and imbalanced sequencing coverage. Over- or under-estimation of gene expression levels will hinder accurate gene differential expression analysis. Here we evaluated strategies to identify expression differences of genes with high coverage in bacterial transcriptome data using either raw sequence reads or unique reads with duplicate fragments removed. In addition, we proposed a generalised linear model (GLM) based approach to identify imbalance in read coverage based on sequence compositions. Our results show that analysis using raw reads identifies more differentially expressed genes with more accurate fold change than using unique reads. We also demonstrate the presence of sequence composition related biases that are independent of gene expression levels and experimental conditions. Finally, genes that still show strong coverage imbalance after correction were tagged using statistical approach.