Gregory J. Bancroft

Development of Signature-Tagged Mutagenesis in Burkholderia pseudomallei To Identify Genes Important in Survival and Pathogenesis

ABSTRACT Burkholderia pseudomallei, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in recA involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine, p-aminobenzoic acid, and a mutant with an insertion in aroB causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the aroB mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms.

Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly+ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly+ rBCG (DeltaureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. DeltaureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of DeltaureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

THE ROLE OF NATURAL KILLER CELLS IN INNATE RESISTANCE TO INFECTION

Natural killer cells were first identified by their cytotoxic activity against tumor cells, suggesting a role in immunological surveillance against neoplasia. However, there is now increasing evidence that natural killer cells are important mediators of innate resistance against a variety of pathogenic micro-organisms. Recently, several important advances have been made in our understanding of how these cells are activated during infection, the contribution of cytokines derived from natural killer cells to host resistance and their influence on the development of antigen-specific T-cell responses.

Natural Immunity: A T‐Cell‐Independent Pathway of Macrophage Activation, Defined in the scid Mouse

In this review we summarize our studies on macrophage activation in mice with the scid mutation. Such mice exhibit, to a remarkable extent, a cellular system capable of activating macrophages. This pathway of macrophage activation defmes the cellular immune system operating presumably in its most primitive form without the modulation of B or T cells, i.e., without adaptive immunity. This T cell-independent mechanism of macrophage activation constitutes an important line of defense and protection against certain microbes in scid mice. It is likely that the T cell-independent pathway is also part of the normal response of mice with an intact immune system. Because it is not masked by the T-cell response, the JctV/mouse is an excellent model to defme the cellular and chemical components of the T cell-independent pathway. To date, we have defmed two cell types operating in this system, namely macrophages and natural killer (NK) cells, and three cytokines, interferon-y (IFNy), tumor necrosis factor (TNF) and interleukin-1 (IL-1). Our review summarizes our published results plus those of studies now in press or submitted (Bancroft et al. 1986,1987,1989a, 1989b, DeSchryver-Kecskemetietal. 1988, Wherry etal. 1991, Rogers et al. 1991, Bancroft & Webster 1991).

Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens.

The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis. In vivo experiments in mice have identified that a rapid IFN-γ response is essential for host survival. To identify the cellular sources of IFN-γ, spleen cells from uninfected mice were stimulated with B. pseudomallei in vitro and assayed by ELISA and flow cytometry. Costaining for intracellular IFN-γ vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8+ T cells were the dominant sources of IFN-γ. IFN-γ+ NK cells were detectable after 5 h and IFN-γ+ CD8+ T cells within 15 h after addition of bacteria. IFN-γ production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect. Three-color flow cytometry showed that IFN-γ-producing CD8+ T cells were of the CD44high phenotype. The preferential activation of NK cells and CD8+ T cells, rather than CD4+ T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo. This rapid mechanism of CD8+ T cell activation may be an important component of innate immunity to intracellular pathogens.

Deletion of two-component regulatory systems increases the virulence of Mycobacterium tuberculosis.

ABSTRACT Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors. We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain. The most hypervirulent mutant (devRΔ) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that of the host.

Characterization of Auxotrophic Mutants of Mycobacterium tuberculosis and Their Potential as Vaccine Candidates

ABSTRACT Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and themetB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpDmutants were killed during this time. In SCID mice, both H37Rv and themetB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. TheproC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and themetB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proCand trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either theproC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.

Enhanced protection to Mycobacterium tuberculosis infection in IL-10-deficient mice is accompanied by early and enhanced Th1 responses in the lung

IL‐10 regulates the balance of an immune response between pathogen clearance and immunopathology. We show here that Mycobacterium tuberculosis (Mtb) infection in the absence of IL‐10 (IL‐10−/− mice) results in reduced bacterial loads in the lung. This reduction was preceded by an accelerated and enhanced IFN‐γ response in the lung, an increased influx of CD4+ T cells into the lung, and enhanced production of chemokines and cytokines, including CXCL10 and IL‐17, in both the lung and the serum. Neutralization of IL‐17 affected neither the enhanced production of CXCL10 nor the accumulation of IFN‐γ‐producing T cells in the lungs, but led to reduced numbers of granulocytes in the lung and reduced bacterial loads in the spleens of Mtb‐infected mice. This suggests that IL‐17 may contribute to dissemination of Mtb.

Optimisation of bioluminescent reporters for use with mycobacteria.

Background Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI. Methodology/Principal Findings One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging. Conclusions/Significance While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

Production of Matrix Metalloproteinases in Response to Mycobacterial Infection

ABSTRACT Matrix metalloproteinases (MMPs) constitute a large family of enzymes with specificity for the various proteins of the extracellular matrix which are implicated in tissue remodeling processes and chronic inflammatory conditions. To investigate the role of MMPs in immunity to mycobacterial infections, we incubated murine peritoneal macrophages with viable Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv and assayed MMP activity in the supernatants by zymography. Resting macrophages secreted only small amounts of MMP-9 (gelatinase B), but secretion increased dramatically in a dose-dependent manner in response to either BCG or M. tuberculosis in vitro. Incubation with mycobacteria also induced increased MMP-2 (gelatinase A) activity. Neutralization of tumor necrosis alpha (TNF-α), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria. Exogenous addition of TNF-α or IL-18 induced macrophages to express MMPs, even in the absence of bacteria. The immunoregulatory cytokines gamma interferon (IFN-γ), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms. IFN-γ treatment increased macrophage secretion of TNF-α but still reduced their MMP activity. Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-α available to the macrophages. Finally, infection of BALB/c or severe combined immunodeficiency (SCID) mice with either BCG or M. tuberculosis induced substantial increases in MMP-9 activity in infected tissues. In conclusion, we show that mycobacterial infection induces MMP-9 activity both in vitro and in vivo and that this is regulated by TNF-α, IL-18, and IFN-γ. These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections.