Mary K. Hondalus

Attenuation of and Protection Induced by a Leucine Auxotroph of Mycobacterium tuberculosis

ABSTRACT Attenuated mutants of Mycobacterium tuberculosisrepresent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph of M. tuberculosiswas created by allelic exchange, replacing wild-type leuD(Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was <10−11. In contrast to wild-type M. tuberculosis, the ΔleuD mutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-type leuD abolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the ΔleuD mutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-type M. tuberculosis, were immunized with the ΔleuD mutant and subsequently challenged with virulent M. tuberculosisby both the intravenous and aerosol routes. A comparison group of mice was immunized with conventional Mycobacterium bovis BCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the ΔleuD mutant of M. tuberculosis exhibited enhanced and statistically equivalent survival curves. However, theleuD auxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection against M. tuberculosis challenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains ofM. tuberculosis should persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.

Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth

Previous work established that the principal sigma factor (RpoV) of virulent Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, restores virulence to an attenuated strain containing a point mutation (Arg-515→His) in the 4.2 domain of RpoV. We used the 4.2 domain of RpoV as bait in a yeast two-hybrid screen of an M. tuberculosis H37Rv library and identified a putative transcription factor, WhiB3, which selectively interacts with the 4.2 domain of RpoV in virulent strains but not with the mutated (Arg-515→His) allele. Infection of mice and guinea pigs with a M. tuberculosis H37Rv whiB3 deletion mutant strain showed that whiB3 is not necessary for in vivo bacterial replication in either animal model. In contrast, an M. bovis whiB3 deletion mutant was completely attenuated for growth in guinea pigs. However, we found that immunocompetent mice infected with the M. tuberculosis H37Rv whiB3 mutant strain had significantly longer mean survival times as compared with mice challenged with wild-type M. tuberculosis. Remarkably, the bacterial organ burdens of both mutant and wild-type infected mice were identical during the acute and persistent phases of infection. Our results imply that M. tuberculosis replication per se is not a sufficient condition for virulence in vivo. They also indicate a different role for M. bovis and M. tuberculosis whiB3 genes in pathogenesis generated in different animal models. We propose that M. tuberculosis WhiB3 functions as a transcription factor regulating genes that influence the immune response of the host.

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.

Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcus equi by activated macrophages.

ABSTRACT Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-γ), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO−), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS−/−) or the oxidative burst (gp91phox−/−) are more susceptible to lethalR. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O2−) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO− efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO− in situ. Inhibition of nitric oxide production by activated macrophages by usingNG-monomethyl-l-arginine blocks their production of ONOO− and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equiby IFN-γ-activated macrophages.

Protection Elicited by a Double Leucine and Pantothenate Auxotroph of Mycobacterium tuberculosis in Guinea Pigs

ABSTRACT We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The ΔleuD ΔpanCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.

Redox homeostasis in mycobacteria: the key to tuberculosis control?

Mycobacterium tuberculosis (Mtb) is a metabolically flexible pathogen that has the extraordinary ability to sense and adapt to the continuously changing host environment experienced during decades of persistent infection. Mtb is continually exposed to endogenous reactive oxygen species (ROS) as part of normal aerobic respiration, as well as exogenous ROS and reactive nitrogen species (RNS) generated by the host immune system in response to infection. The magnitude of tuberculosis (TB) disease is further amplified by exposure to xenobiotics from the environment such as cigarette smoke and air pollution, causing disruption of the intracellular prooxidant–antioxidant balance. Both oxidative and reductive stresses induce redox cascades that alter Mtb signal transduction, DNA and RNA synthesis, protein synthesis and antimycobacterial drug resistance. As reviewed in this article, Mtb has evolved specific mechanisms to protect itself against endogenously produced oxidants, as well as defend against host and environmental oxidants and reductants found specifically within the microenvironments of the lung. Maintaining an appropriate redox balance is critical to the clinical outcome because several antimycobacterial prodrugs are only effective upon bioreductive activation. Proper homeostasis of oxido-reductive systems is essential for Mtb survival, persistence and subsequent reactivation. The progress and remaining deficiencies in understanding Mtb redox homeostasis are also discussed.

Diagnosis, Treatment, Control, and Prevention of Infections Caused by Rhodococcus equi in Foals

Rhodococcus equi, a gram-positive facultative intracellular pathogen, is one of the most common causes of pneumonia in foals. Although R. equi can be cultured from the environment of virtually all horse farms, the clinical disease in foals is endemic at some farms, sporadic at others, and unrecognized at many. On farms where the disease is endemic, costs associated with morbidity and mortality attributable to R. equi may be very high. The purpose of this consensus statement is to provide recommendations regarding the diagnosis, treatment, control, and prevention of infections caused by R. equi in foals.

Rhodococcus equi: Clinical Manifestations, Virulence, and Immunity

Pneumonia is a major cause of disease and death in foals. Rhodococcus equi, a gram-positive facultative intracellular pathogen, is a common cause of pneumonia in foals. This article reviews the clinical manifestations of infection caused by R. equi in foals and summarizes current knowledge regarding mechanisms of virulence of, and immunity to, R. equi. A complementary consensus statement providing recommendations for the diagnosis, treatment, control, and prevention of infections caused by R. equi in foals can be found in the same issue of the Journal.

Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

Innate Immune Responses to Rhodococcus equi

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-κB and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-KB and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2−/− mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2−/−-deficient but not TLR4−/−-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.