Iron-sparing Response of Mycobacterium avium subsp. paratuberculosis is strain dependent

<p>Abstract</p> <p>Background</p> <p>Two genotypically and microbiologically distinct strains of <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) exist - S and C MAP strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributing factor for the observed microbiologic differences. We recently demonstrated that S strains have defective iron storage systems, leading us to propose that these strains might experience iron toxicity when excess iron is provided in the medium. To test this hypothesis, we carried out transcriptional and proteomic profiling of these MAP strains under iron-replete or -deplete conditions.</p> <p>Results</p> <p>We first complemented <it>M. smegmatis</it>Δ<it>ideR </it>with IdeR of C MAP or that derived from S MAP and compared their transcription profiles using <it>M. smegmatis mc</it>^<it>2</it><it>155 </it>microarrays. In the presence of iron, sIdeR repressed expression of <it>bfrA </it>and MAP2073c, a ferritin domain containing protein suggesting that transcriptional control of iron storage may be defective in S strain. We next performed transcriptional and proteomic profiling of the two strain types of MAP under iron-deplete and -replete conditions. Under iron-replete conditions, C strain upregulated iron storage (BfrA), virulence associated (Esx-5 and antigen85 complex), and ribosomal proteins. In striking contrast, S strain downregulated these proteins under iron-replete conditions. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation resulted in the identification of four unannotated proteins. Two of these were upregulated by a C MAP strain in response to iron supplementation. The iron-sparing response to iron limitation was unique to the C strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division).</p> <p>Conclusions</p> <p>Taken together, our study revealed that C and S strains of MAP utilize divergent metabolic pathways to accommodate in vitro iron stress. The knowledge of the metabolic pathways these divergent responses play a role in are important to 1) advance our ability to culture the two different strains of MAP efficiently, 2) aid in diagnosis and control of Johne's disease, and 3) advance our understanding of MAP virulence.</p

Frontline Science: Employing enzymatic treatment options for management of ocular biofilm-based infections

Pseudomonas aeruginosa-induced corneal keratitis is a sight-threatening disease. The rise of antibiotic resistance among P. aeruginosa keratitis isolates makes treatment of this disease challenging, emphasizing the need for alternative therapeutic modalities. By comparing the responses to P. aeruginosa infection between an outbred mouse strain (Swiss Webster, SW) and a susceptible mouse strain (C57BL6/N), we found that the inherent neutrophil-killing abilities of these strains correlated with their susceptibility to infection. Namely, SW-derived neutrophils were significantly more efficient at killing P. aeruginosa in vitro than C57BL6/N-derived neutrophils. To interrogate whether the distinct neutrophil killing capacities were dependent on endogenous or exogenous factors, neutrophil progenitor cell lines were generated. The in vitro differentiated neutrophils from either SW or C57BL6/N progenitors retained the differential killing abilities, illustrating that endogenous factors conferred resistance. Consistently, quantitative LC-MS/MS analysis revealed strain-specific and infection-induced alterations of neutrophil proteomes. Among the distinctly elevated proteins in the SW-derived proteomes were alpha-mannosidases, potentially associated with protection. Inhibition of alpha-mannosidases reduced neutrophil bactericidal functions in vitro. Conversely, topical application of alpha-mannosidases reduced bacterial biofilms and burden of infected corneas. Cumulatively, these data suggest novel therapeutic approaches to control bacterial biofilm assembly and improve bacterial clearance via enzymatic treatments

Role of Microbiota in Strengthening Ocular Mucosal Barrier Function Through Secretory IgA

PURPOSE. The purpose of this study was to evaluate mechanisms controlling secretory IgA (SIgA) production, thereby ensuring maintenance of ocular surface health. METHODS. To determine whether the presence of specific gut commensal species regulates SIgA levels and IgA transcripts in the eye-associated lymphoid tissues (EALT), specificpathogen- free (SPF) Swiss Webster (SW) mice were treated with antibiotic cocktails, germfree (GF) SW mice were reconstituted with diverse commensal gut microbiota, or monocolonized with gut-specific commensals. Proteomic profiling and quantitative real-time polymerase chain reaction (qRT-PCR) were used to quantify SIgA and IgA levels. 16S rDNA sequencing was carried out to characterize commensal microbiota. RESULTS. Commensal presence regulated ocular surface SIgA levels and mRNA IgA transcripts in EALT. Oral antibiotic cocktail intake significantly reduced gut commensal presence, while maintaining ocular surface commensal levels reduced SIgA and IgA transcripts in EALT. Analysis of gut microbial communities revealed that SPF SW mice carried abundant Bacteroides organisms when compared to SPF C57BL6/N mice, with B. acidifaciens being the most prominent species in SPF SW mice. Monocolonization of GF SW mice with B. acidifaciens, a strict gut anaerobe, resulted in significant increase of IgA transcripts in the EALT, implying generation of B-cell memory. CONCLUSIONS. These data illustrated a "gut-eye'' axis of immune regulation. Exposure of the host to gut commensal species may serve as a priming signal to generate B-cell repertoires at sites different from the gut, such as EALT, thereby ensuring broad protection

A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted

Role of carriers in the transmission of pneumonia in bighorn sheep (Ovis canadensis)

In the absence of livestock contact, recurring lamb mortality in bighorn sheep (Ovis canadensis) populations previously exposed to pneumonia indicates the likely presence of carriers of pneumonia-causing pathogens, and possibly inadequate maternally derived immunity. To investigate this problem we commingled naïve, pregnant ewes (n=3) with previously exposed rams (n=2). Post-commingling, all ewes and lambs born to them acquired pneumonia-causing pathogens (leukotoxin-producing Pasteurellaceae and Mycoplasma ovipneumoniae), with subsequent lamb mortality between 4-9 weeks of age. Infected ewes became carriers for two subsequent years and lambs born to them succumbed to pneumonia. In another experiment, we attempted to suppress the carriage of leukotoxin-producing Pasteurellaceae by administering an antibiotic to carrier ewes, and evaluated lamb survival. Lambs born to both treatment and control ewes (n=4 each) acquired pneumonia and died. Antibody titers against leukotoxin-producing Pasteurellaceae in all eight ewes were ‘protective’ (>1:800 and no apparent respiratory disease); however their lambs were either born with comparatively low titers, or with high (but non-protective) titers that declined rapidly within 2-8 weeks of age, rendering them susceptible to fatal disease. Thus, exposure to pneumonia-causing pathogens from carrier ewes, and inadequate titers of maternally derived protective antibodies, are likely to render bighorn lambs susceptible to fatal pneumonia

A Multivalent Mannheimia-Bibersteinia Vaccine Protects Bighorn Sheep against Mannheimia haemolytica Challenge

Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia - Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 × 10 5 CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia