Unsaturated Fatty Acids Promote the Phagocytosis of P. aeruginosa and R. equi by RAW264.7 Macrophages

In the present study, using the murine monocyte/macrophage cell line RAW264.7 as a model system, we analyzed the phagocytosis rate and the bactericidal capacity of polyunsaturated fatty acids (PUFA)-enriched macrophages against Pseudomonas aeruginosa and Rhodococcus equi. The P. aeruginosa strain ATCC 10145, the virulent R. equi strain ATCC 33701, and the non-virulent R. equi strain ATCC 6939 were examined. Flow cytometric detection of intracellular microorganisms in combination with viability assays were used to determine the impact of PUFA on the number of engulfed, surviving as well as replicating bacteria. Macrophage enrichment with PUFA resulted in an increase of the internalization rate of the microorganisms by the immune cells. Moreover, an impeding action of the unsaturated fatty acids on the intracellular survival rates of the virulent strains P. aeruginosa ATCC 10145 and R. equi ATCC 33701 could be observed. The n-3 fatty acid docosahexaenoic acid (DHA) as well as the n-6 fatty acid arachidonic acid (AA) showed the most pronounced effects. Taken together, our data support the idea of supplementing PUFA to immunocompromised individuals as well as to people suffering from chronic infections with P. aeruginosa or R. equi to improve macrophage phagocytic and microbicidal activity.     

Conjugal Transfer of a Virulence Plasmid in the Opportunistic Intracellular Actinomycete Rhodococcus equi

Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ∼81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.     

Associations between the Ecology of Virulent Rhodococcus equi and the Epidemiology of R. equi Pneumonia on Australian Thoroughbred Farms

The ecology of virulent strains of Rhodococcus equi on horse farms is likely to influence the prevalence and severity of R. equi pneumonia in foals. This study examined the association between the ecology of virulent R. equi and the epidemiology of R. equi pneumonia by collecting air and soil samples over two breeding seasons (28 farm-year combinations) on Thoroughbred breeding farms with different reported prevalences of R. equi pneumonia. Colony blotting and DNA hybridization were used to detect and measure concentrations of virulent R. equi. The prevalence of R. equi pneumonia was associated with the airborne burden of virulent R. equi (both the concentration and the proportion of R. equi bacteria that were virulent) but was not associated with the burden of virulent R. equi in the soil. Univariable screening and multivariable model building were used to evaluate the effect of environmental and management factors on virulent R. equi burdens. Lower soil moisture concentrations and lower pasture heights were significantly associated with elevated airborne concentrations of virulent R. equi, as were the holding pens and lanes, which typically were sandy, dry, and devoid of pasture cover. Few variables appeared to influence concentrations of virulent R. equi in soil. Acidic soil conditions may have contributed to an elevated proportion of virulent strains within the R. equi population. Environmental management strategies that aim to reduce the level of exposure of susceptible foals to airborne virulent R. equi are most likely to reduce the impact of R. equi pneumonia on endemically affected farms.     

Intracellular replication of attenuated Mycobacterium tuberculosis phoP mutant in the absence of host cell cytotoxicity

Intracellular pathogen Mycobacterium tuberculosis survives and replicates in macrophages but limited information is available on its replication into non-phagocytic cells. Here we study the role of the M. tuberculosis virulence gene phoP in the intracellular growth with rat and human lung fibroblasts. In contrast to macrophages, attenuated M. tuberculosis phoP mutant was able to multiply intracellularly in fibroblasts at the same level as the virulent M. tuberculosis. However, when M. tuberculosis virulence was studied using human foetal lung fibroblasts, MRC-5 cell line, the virulent strain caused a significant damage in cells compared with attenuated strains BCG and M. tuberculosis phoP mutant. We analysed the effect of cytoskeleton inhibitors in NRK-49F fibroblasts. M. tuberculosis invasion was not inhibited, suggesting that mycobacterial uptake was microtubule and microfilament independent. Our results suggest that PhoP in M. tuberculosis does not regulate intracellular replication in fibroblasts, contrary to what happens in macrophages. The ability of M. tuberculosis phoP mutant to replicate within non-phagocytic cells, such as fibroblasts, without causing damage, could be a potential advantage for a live attenuated vaccine against tuberculosis.     

Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland

Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15–17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85–90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern.     

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10⁶ cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10⁷ cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.     

Mycobacterium tuberculosis induces an atypical cell death mode to escape from infected macrophages

Background

Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds ∼20 per macrophage but the precise nature of this demise has not been defined.

Methodology/Principal Findings

We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system.

Conclusions/Significance

Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis.     

VapA of Rhodococcus equi binds phosphatidic acid

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi‐containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA^32‐189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host‐derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA^32‐189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.     

Virulence‐associated protein A from Rhodococcus equi is an intercompartmental pH‐neutralising virulence factor

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences.     

Versatile <Emphasis Type="Italic">Rhodococcus equi</Emphasis>–<Emphasis Type="Italic">Escherichia coli</Emphasis> shuttle vectors

Rhodococcus equi is an intracellular pathogen of macrophages, causing disease in young foals, humans, and sporadically other animals. Although R. equi is easy to grow and manipulate, the analysis of virulence is hampered by a lack of molecular tools. This paper describes the development of a number of versatile plasmids for use in R. equi. Plasmids pREV2 and pREV5 use origins of replication derived from the Mycobacterium fortuitum plasmids pAL5000 and pMF1. These plasmids and their derivatives are compatible in R. equi, allowing their use for analysis of gene function in trans. The stability of these plasmids in R. equi in the absence of selection for the plasmid borne antibiotic resistance markers, and their integrity following passage through Escherichia coli and R. equi was determined.     

Immunogenicity of an Electron Beam Inactivated Rhodococcus equi Vaccine in Neonatal Foals

Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.     

Pathogenicity of Mycobacterium tuberculosis Is Expressed by Regulating Metabolic Thresholds of the Host Macrophage

The success of Mycobacterium tuberculosis as a pathogen derives from its facile adaptation to the intracellular milieu of human macrophages. To explore this process, we asked whether adaptation also required interference with the metabolic machinery of the host cell. Temporal profiling of the metabolic flux, in cells infected with differently virulent mycobacterial strains, confirmed that this was indeed the case. Subsequent analysis identified the core subset of host reactions that were targeted. It also elucidated that the goal of regulation was to integrate pathways facilitating macrophage survival, with those promoting mycobacterial sustenance. Intriguingly, this synthesis then provided an axis where both host- and pathogen-derived factors converged to define determinants of pathogenicity. Consequently, whereas the requirement for macrophage survival sensitized TB susceptibility to the glycemic status of the individual, mediation by pathogen ensured that the virulence properties of the infecting strain also contributed towards the resulting pathology.     

Attenuated Mycobacterium tuberculosis SO2 Vaccine Candidate Is Unable to Induce Cell Death

It has been proposed that Mycobacterium tuberculosis virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the M. tuberculosis phoP mutant SO2, we sought to evaluate its potential to induce host cell death. The parental M. tuberculosis MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG) were used as comparators in mouse models in vitro and in vivo. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages in vitro nor during lung infection in vivo. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during M. tuberculosis infection.     

A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated ΔsupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the ΔsupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive.     

Characterization of Burkholderia pseudomallei Strains Using a Murine Intraperitoneal Infection Model and In Vitro Macrophage Assays

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a gram-negative facultative intracellular bacterium. This bacterium is endemic in Southeast Asia and Northern Australia and can infect humans and animals by several routes. It has also been estimated to present a considerable risk as a potential biothreat agent. There are currently no effective vaccines for B. pseudomallei, and antibiotic treatment can be hampered by nonspecific symptomology, the high incidence of naturally occurring antibiotic resistant strains, and disease chronicity. Accordingly, there is a concerted effort to better characterize B. pseudomallei and its associated disease. Before novel vaccines and therapeutics can be tested in vivo, a well characterized animal model is essential. Previous work has indicated that mice may be a useful animal model. In order to develop standardized animal models of melioidosis, different strains of bacteria must be isolated, propagated, and characterized. Using a murine intraperitoneal (IP) infection model, we tested the virulence of 11 B. pseudomallei strains. The IP route offers a reproducible way to rank virulence that can be readily reproduced by other laboratories. This infection route is also useful in distinguishing significant differences in strain virulence that may be masked by the exquisite susceptibility associated with other routes of infection (e.g., inhalational). Additionally, there were several pathologic lesions observed in mice following IP infection. These included varisized abscesses in the spleen, liver, and haired skin. This model indicated that commonly used laboratory strains of B. pseudomallei (i.e., K96243 and 1026b) were significantly less virulent as compared to more recently acquired clinical isolates. Additionally, we characterized in vitro strain-associated differences in virulence for macrophages and described a potential inverse relationship between virulence in the IP mouse model of some strains and in the macrophage phagocytosis assay. Strains which were more virulent for mice (e.g., HBPU10304a) were often less virulent in the macrophage assays, as determined by several parameters such as intracellular bacterial replication and host cell cytotoxicity.     

Study of Lysozyme Resistance in Rhodococcus equi

Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.     

Interactions Between Macrophages of Guinea Pigs and Salmonellae I. Fate of Salmonella typhimurium Within Macrophages of Normal Guinea Pigs

A suspended cell culture procedure was described for the cultivation of guinea pig macrophages infected with Salmonella typhimurium. The fate of the intracellular bacteria was assessed by quantitative recovery of viable bacteria with 0.5% solution of sodium desoxycholate. Two strains of S. typhimurium with different degrees of virulence for mice were compared. There was an initial destruction of intracellular bacteria of both strains; however, the extent of this destruction differed. Approximately 1% of the avirulent bacteria initially phagocytized survived at the end of 4 hr, whereas approximately 8% of the virulent bacteria survived at the end of 3 hr. After this initial killing, the intracellular bacteria began to multiply at a logarithmic rate between 3 and 21 hr after phagocytosis, and then a stationary phase was attained. The rate of this multiplication was comparable for both strains.     

The Balance of Apoptotic and Necrotic Cell Death in Mycobacterium tuberculosis Infected Macrophages Is Not Dependent on Bacterial Virulence

Background

An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission.

Methods

We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237.

Results

We show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis – both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis.

Conclusions

This reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.     

Deletion of vapA encoding Virulence Associated Protein A attenuates the intracellular actinomycete Rhodococcus equi

Virulent strains of the facultative intracellular bacterium Rhodococcus equi isolated from young horses (foals) with R. equi pneumonia, carry an 80-90 kb virulence plasmid and express a highly immunogenic 15-17 kDa protein of unknown function called VapA (Virulence Associated Protein A). Recent sequencing of the virulence plasmid identified a putative pathogenicity island encoding a novel family of seven Vap proteins including VapA. These proteins exhibit a significant sequence similarity to each other but have no homologues in other organisms. In this study, we describe the construction of an R. equi mutant lacking a 7.9 kb DNA region spanning five vap genes (vapA, -C, -D, -E and -F ). This vap locus mutant was attenuated for virulence in mice as it was unable to replicate in vivo and was rapidly cleared in comparison to the virulent wild-type strain. Complementation analysis of the vap locus mutant showed that expression of vapA alone could restore full virulence, whereas expression of vapC, -D and -E could not. We subsequently constructed an R. equi strain lacking only the vapA gene and found that it was attenuated for growth in vivo to the same degree as the vap locus mutant. Unlike wild-type R. equi which replicates intracellularly, both of the mutant strains exhibited a growth defect in macrophages although their attachment to the macrophages was unaffected. These studies provide the first proof of a role for vapA in the virulence of R. equi, and demonstrate that its presence is essential for intracellular growth in macrophages.