No abstract

In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the transcriptional regulator DosR, resulting in the induction of the DosR regulon, which is important for anaerobic survival and perhaps latent infection. The individual and collective roles of these sensors have been postulated biochemically, but their roles in vivo have remained unclear. This work demonstrates distinct and additive roles for each sensor during anaerobic dormancy. Both sensors are necessary for wild-type levels of DosR regulon induction, and concomitantly, full induction of the regulon is required for wild-type anaerobic survival. In the anaerobic model, DosT plays an early role, responding to hypoxia. DosT then induces the regulon and with it DosS, which sustains and further induces the regulon. DosT then loses its functionality as oxygen becomes limited, and DosS alone maintains induction of the genes from that point forward. Thus, M. tuberculosis has evolved a system whereby it responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic state.

The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.Mycobacteria are strict aerobes, but Mycobacterium tuberculosis encounters microaerobic to anaerobic environments during the course of infection. Oxygen-limited microenvironments occur in mature granulomas, which are known to be avascular, inflammatory, and necrotic (1,5,20,40,56). Recent reports have detected mycobacterial DNA in visibly normal lung tissue (17), as well as adipose tissue (32). Interestingly, adipose tissue has been associated with hypoxia as well as the presence of nitric oxide (NO), both of which inhibit respiration (38,51,57,60). Further, M. tuberculosis is able to survive for long periods of time in a nonreplicating and nonrespiring state (15, 55).The DosR regulon is expressed in response to hypoxia, NO, and carbon monoxide (CO) and is thought to be important for early adaptation to these stimuli as well as long-term survival in the host (2,25,26,46,47,53,58). Despite induction by other gases, the presence of oxygen itself inhibits induction of the regulon (26,41,43,48). The DosR regulon is regulated by the response regulator DosR (DevR; Rv3133c) and was recently shown to be positively regulated by PhoP (Rv0757) to a basal level during aerobic growth (16). DosR is activated by two sensor histidine kinases, DosT (Rv2027c) and DosS (DevS; Rv3132c). The activation of DosR occurs through autophosphorylatio...

The role of gut microbiomes as important regulators of mammalian health is increasingly recognized, although feline and canine gut microbiomes remain poorly characterized. In this proof-of-concept study, we assessed the utility of a direct-to-consumer approach to executing pet microbiome studies. We characterized the gut microbiomes of 238 pets (46 cats and 192 dogs) by generating~11 million merged reads that were mapped to the V4 region of 16S ribosomal RNA gene at a sequencing depth of 45,806 (±22,325) reads per sample. Analyses of these reads revealed that both feline and canine gut microbiomes are dominated by three major phyla, namely Firmicutes, Proteobacteria, and Bacteroides and that alpha diversity is higher in the feline gut. In addition to interspecies differences between the feline and canine gut, we also detected appreciable intraspecies bacterial variation within the canine population. While the dogs in this dataset could be assigned to three distinct clusters based on their gut microbiome, no clustering was observed within the feline population. Integration of additional data obtained from survey questionnaires revealed that geography and body weight may be associated with canine gut microbiome composition. Furthermore, we found that both the inter and intraspecies differences are more pronounced at finer taxonomic levels, indicating that strain-level investigations may be necessary in the future. This study demonstrates that the direct-to-consumer approach overcomes existing limitations in pet microbiome research, for example, it allows collection of large numbers of pet samples. The direct-to-consumer approach has proven successful in human genomics as well as human microbiomics and this study demonstrates that by building partnerships with an engaged general public this approach can also propel the field of pet microbiomics forward.

Since the discovery of antibiotics, mortality due to bacterial infection has decreased dramatically. However, infections from difficult to treat bacteria such as Mycobacterium tuberculosis and multidrug-resistant pathogens have been on the rise. An understanding of the cascade of events that leads to cell death downstream of specific drug-target interactions is not well understood. We have discovered that killing by several classes of antibiotics was stopped by maintaining pH balance within the bacterial cell, consistent with a shared mechanism of antibiotic killing. Our findings suggest a mechanism of antibiotic killing that stems from the antibiotic’s ability to increase the pH within bacterial cells by disrupting proton entry without affecting proton pumping out of cells. Knowledge of the core mechanism necessary for antibiotic killing could have a significant impact on the development of new lethal antibiotics and for the treatment of recalcitrant and drug-resistant pathogens.

Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo. Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740), a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one-third of the world's population. The sole extant vaccine for tuberculosis is the live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG). We examined 13 representative BCG strains from around the world to ascertain their ability to express DosR-regulated dormancy antigens. These are known to be recognized by T cells of M. tuberculosis-infected individuals, especially those harboring latent infections. Differences in the expression of these antigens could be valuable for use as diagnostic markers to distinguish BCG vaccination from latent tuberculosis. We determined that all BCG strains were defective for the induction of two dormancy genes: narK2 (Rv1737c) and narX (Rv1736c). NarK2 is known to be necessary for nitrate respiration during anaerobic dormancy. Analysis of the narK2/X promoter region revealed a base substitution mutation in all tested BCG strains and M. bovis in comparison to the M. tuberculosis sequence. We also show that nitrate reduction by BCG strains during dormancy was greatly reduced compared to M. tuberculosis and varied between tested strains. Several dormancy regulon transcriptional differences were also identified among the strains, as well as variation in their growth and survival. These findings demonstrate defects in DosR regulon expression during dormancy and phenotypic variation between commonly used BCG vaccine strains.

Background Probiotics have been demonstrated to ameliorate clinical signs of gastrointestinal diseases in dogs in various studies. However, the effect of probiotics in a healthy population, as well as factors contributing individualized responses, remain largely unknown. This trial examined gut microbiota (GM) and health outcomes in household dogs after synbiotic (SN) supplementation containing probiotics and inulin (a prebiotic). Healthy dogs were randomized to receive SN (50 mg/d inulin and 20 billion total CFU/d of L. reuteri, P. acidilactici, E. faecium, L. acidophilus, B. animalis, L. fermentum, L. rhamnosus) or placebo (PL) for 4 weeks. Owners completed a health survey and collected stool samples for GM profiling (shotgun metagenomic sequencing) at baseline and week 4 in both groups, and at week 6 in the SN group. Results A significant shift (p < 0.001) in β-diversity was observed in the SN (n = 24), but not PL group (n = 19), at week 4 relative to baseline. Forty-five bacterial species, 43 (96%) of which were Lactobacillales, showed an increase in the relative abundances (≥2 fold change, adjusted p < 0.05) in the SN group at week 4. E. coli also decreased at week 4 in the SN group (2.8-fold, adjusted p < 0.01). The altered taxa largely returned to baseline at week 6. The degree of changes in β-diversity was associated with GM at baseline. Specifically, dogs with higher Proteobacteria and lower Lactobacillales responded more robustly to supplementation in terms of the change in β-diversity. Dogs fed SN tended to have lower diarrhea incidence (0% vs 16%, p = 0.08). Conclusions SN supplement had a short-term impact on the gut microbiota in healthy household dogs as characterized by shotgun metagenomic sequencing. Findings warrant further investigation with longer duration and populations at risk of gastrointestinal diseases. The magnitude of response to the supplement was associated with microbial profile at baseline. To our knowledge, this is the first study documenting such association and may provide a basis for personalized nutrition in companion dogs.