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

Neutrophil L-plastin controls ocular paucibacteriality and susceptibility to keratitis

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1), an actin bundling protein, resulted in an ocular commensal overgrowth, characterized with increased presence of conjunctiva

Growth of Mannheimia haemolytica:Inhibitory agents and putative mechanism of inhibition

Pneumonia is the most important disease of bighorn sheep (BHS). Pathogens detected in pneumonic BHS lungs include Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida and Mycoplasma ovipneumoniae. Although leukotoxin-producing M. haemolytica consistently causes fatal pneumonia in BHS under experimental conditions, B. trehalosi and P. multocida are isolated more frequently than M. haemolytica from pneumonic BHS lungs by culture. In this study, I extended the previous findings from our laboratory that B. trehalosi and P. multocida inhibit the growth of M. haemolytica. I hypothesized that the inhibitory phenotype of B. trehalosi is conserved across B. trehalosi strains. Fifty-five B. trehalosi isolates were tested by bacterial competition assays. All of them inhibited M. haemolytica suggesting that the inhibitory phenotype is conserved. No plasmids were isolated from any of the 30 B. trehalosi isolates tested, suggesting that the effectors are chromosomally-encoded.The observation that M. haemolytica was not isolated frequently even from pneumonic BHS lungs that did not carry B. trehalosi or P. multocida led to my second hypothesis that bacteria other than B. trehalosi and P. multocida inhibit the growth of M. haemolytica. Bacterial competition assays showed that Escherichia coli also inhibits the growth of M. haemolytica via a proximity-dependent mechanism. Streptococcus spp. and Staphylococcus spp. did not have any inhibitory effect.Fatal pneumonia in BHS often results from acquisition of leukotoxin-positive M. haemolytica from domestic sheep (DS). I hypothesized that intranasal inoculation of B. trehalosi will eliminate or reduce shedding of M. haemolytica by DS. Domestic sheep were oro-nasally inoculated with B. trehalosi following antibiotic treatment. These DS shed reduced numbers of M. haemolytica than un-treated DS. However, healthy BHS commingled with B. trehalosi-treated DS acquired M. haemolytica around day 14 post-commingling which resulted in the development of leukotoxin-neutralizing antibodies. They did not develop pneumonia for 211 days. However, they died of pneumonia when commingled with DS that carried leukotoxin-positive M. haemolytica, B. trehalosi and M. ovipneumoniae. These findings suggest that B. trehalosi can be used to reduce the shedding of M. haemolytica by DS. However, infection with leukotoxin-positive M. haemolytica, B. trehalosi and M. ovipneumoniae synergistically causes fatal pneumonia in BHS

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

β-Hemolysis May Not Be a Reliable Indicator of Leukotoxicity of Mannheimia haemolytica Isolates

Mannheimia (Pasteurella) haemolytica causes bronchopneumonia in domestic and wild ruminants. Leukotoxin is the critical virulence factor of M. haemolytica. Since &beta;-hemolysis is caused by a large number of leukotoxin-positive M. haemolytica isolates, all &beta;-hemolytic M. haemolytica isolates are considered to be leukotoxic as well. However, conflicting reports exist in literature as to the leukotoxic and hemolytic properties of M. haemolytica. One group of researchers reported their leukotoxin-deletion mutants to be hemolytic while another reported their mutants to be non-hemolytic. The objective of this study was to determine whether &beta;-hemolysis is a reliable indicator of leukotoxicity of M. haemolytica isolates. Ninety-five isolates of M. haemolytica were first confirmed for presence of leukotoxin gene (lktA) by a leukotoxin-specific PCR assay. Culture supernatant fluids from these isolates were then tested for presence of leukotoxin protein by an ELISA, and for leukotoxic activity by a cytotoxicity assay. All isolates were tested for &beta;-hemolysis by culture on blood agar plates. Sixty-two isolates (65%) produced leukotoxin protein while 33 isolates (35%) did not. Surprisingly, 18 of the 33 isolates (55%), that did not produce leukotoxin protein, were hemolytic. Of the 62 isolates that produced leukotoxin, 55 (89%) were leukotoxic while 7 (11%) were not. All except one of the 55 leukotoxic isolates (98%) were also hemolytic. All seven isolates that were not leukotoxic were hemolytic. Taken together, these results suggest that &beta;-hemolysis may not be a reliable indicator of leukotoxicity of M. haemolytica isolates. Furthermore, all M. haemolytica isolates that possess lktA gene may not secrete active leukotoxin

Proximity-Dependent Inhibition of Growth of Mannheimia haemolytica by Pasteurella multocida

Mannheimia haemolytica , Pasteurella multocida , and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis ). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica . Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica . The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida - M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS

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