Suat L. G. Cirillo

Identification of novel loci involved in entry by Legionella pneumophila.

Legionella pneumophila is primarily an intracellular pathogen during infection; thus, the mechanisms of entry into host cells are likely to be important for pathogenesis. Several L. pneumophila mutants that display an enhanced-entry (Enh) phenotype were isolated by selecting for bacteria that enter host cells at a higher frequency than wild-type. In the course of characterizing the genetic basis of one of these mutants, C3, a strategy was developed for the isolation of laboratory-media-repressed virulence determinants from L. pneumophila. Screens for dominant mutations using a genomic DNA library from C3 resulted in the isolation of three cosmids that confer an Enh phenotype to wild-type L. pneumophila. Transposon mutagenesis of these cosmids allowed identification of three loci that affect entry. Analysis of the putative proteins encoded by these loci, designated rtxA and enhC, demonstrated similarity to repeats in the structural toxin protein and the secreted Sel-1 protein from Caenorhabditis elegans, respectively. L. pneumophila rtxA and enhC mutants display significantly reduced entry into host cells, compared to wild-type bacteria. The phenotype that the cosmids containing these loci confer is most likely due to elevated expression resulting from their presence on multicopy vectors. The use of increased gene copy number to overexpress genes that are normally repressed under laboratory growth conditions is generally applicable to the isolation of virulence determinants from L. pneumophila and other bacterial pathogens.

Imaging tuberculosis with endogenous β-lactamase reporter enzyme fluorescence in live mice

The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for β-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 ± 2 × 102 colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 104 CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus Calmette–Guérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.

A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos

Infection of the zebrafish with Mycobacterium marinum is regarded as a well‐established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin‐layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall‐associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.

Identification of a gene that affects the efficiency of host cell infection by Legionella pneumophila in a temperature-dependent fashion

ABSTRACT The ability to infect host cells is critical for the survival and replication of intracellular pathogens in humans. We previously found that many genes involved in the ability of Legionella pneumophila to infect macrophages are not expressed efficiently under standard laboratory growth conditions. We have developed an approach using expression of L. pneumophila genes from an exogenous constitutive promoter on a low-copy-number vector that allows identification of genes involved in host cell infection. Through the use of this strategy, we found that expression of a gene, lvhB2, enhances the efficiency of L. pneumophila infection of mammalian cells. The putative protein encoded by lvhB2 has similarity to structural pilin subunits of type IV secretion systems. We confirmed that this gene plays a role in host cell infection by the construction of an in-frame deletion in the L. pneumophila lvhB2 gene and complementation of this mutant with the wild-type gene. The lvhB2 mutant does not display a very obvious defect in interactions with host cells when the bacteria are grown at 37°C, but it has an approximately 100-fold effect on entry and intracellular replication when grown at 30°C. These data suggest that lvhB2 plays an important role in the efficiency of host cell infection by L. pneumophila grown at lower temperatures.

Protection of Mycobacterium tuberculosis from Reactive Oxygen Species Conferred by the mel2 Locus Impacts Persistence and Dissemination

ABSTRACT Persistence of Mycobacterium tuberculosis in humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus in M. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of the mel2 locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with the mel2 mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of the mel2 mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox−/− and iNOS−/− mice and bone marrow-derived macrophages suggest that the primary mechanism by which mel2 affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role of mel2 in resistance to ROS is important for persistence and dissemination of M. tuberculosis and suggests that homologues in other bacterial species are likely to play a role in pathogenesis.

Identification of Mycobacterium avium Genes That Affect Invasion of the Intestinal Epithelium

ABSTRACT Invasion of intestinal mucosa of the host by Mycobacterium avium is a critical step in pathogenesis and likely involves several different bacterial proteins, lipids, glycoproteins, and/or glycolipids. Through the screening of an M. avium genomic library in Mycobacterium smegmatis, we have identified a number of M. avium genes that are associated with increased invasion of mucosal epithelial cells. In order to further investigate these genes, we cloned six of them into a plasmid downstream of a strong mycobacterial promoter (L5 mycobacterial phage promoter), resulting in constitutive expression. Bacteria were then evaluated for increased expression and examined for invasion of HT-29 intestinal epithelial cells. The genes identified encode proteins that are similar to (i) M. tuberculosis coenzyme A carboxylase, (ii) M. tuberculosis membrane proteins of unknown function, (iii) M. tuberculosis FadE20, (iv) a Mycobacterium paratuberculosis surface protein, and (v) M. tuberculosis cyclopropane fatty acyl-phopholipid synthase. The constitutive expression of these genes confers to M. avium the ability to invade HT-29 intestinal epithelial cells with a severalfold increase in efficiency compared to both the wild-type M. avium and M. avium containing the vector alone. Using the murine intestinal ligated loop model, it was observed that the constitutive expression of M. avium proteins has a modest impact on the ability to enter the intestinal mucosa when compared with the wild-type control, suggesting that under in vivo conditions these genes are expressed at higher levels. Evaluation of the expression of these invasion-related genes indicated that under conditions similar to the intestinal lumen environment, the genes identified are upregulated. These data suggest that invasion of the intestinal mucosa is an event that requires the participation of several bacterial factors and the expression of the genes that encode them is less observed under standard laboratory growth conditions.

Application of optical imaging to study of extrapulmonary spread by tuberculosis

The incidence of extrapulmonary tuberculosis is increasing, possibly due to the high frequency of co-infection with HIV. Extrapulmonary infections complicate diagnosis, have higher mortality rates and are more difficult to treat. Insight into the mechanisms involved in extrapulmonary spread of tuberculosis is critical to improving management. We set out to better understand extrapulmonary spread kinetics in mice and guinea pigs as well as the effects of infectious dose. We found that extrapulmonary spread occurs at a discrete time point when infected by low-dose aerosol, but at high-dose aerosol it occurs within the first 24h. The ability to follow tuberculosis in real-time during infection would allow us to better address the mechanisms involved. We found that mycobacteria can be optically imaged after pulmonary infection in the mouse lung, suggesting that this technology could be applied to study of extrapulmonary spread of tuberculosis.

Identification of two Mycobacterium marinum loci that affect interactions with macrophages.

ABSTRACT Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel1 and mel2. In silico analyses of the 11 putative genes in these loci suggest that mel1 encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereas mel2 is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly, mel2 is unique to M. marinum and the M. tuberculosis complex and not present in any other sequenced mycobacterial species. M. marinum mutants with mutations in mel1 and mel2, constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes in mel1 and mel2 are important for the ability of M. marinum to infect host cells.

A Mycobacterium marinum mel2 mutant is defective for growth in macrophages that produce reactive oxygen and reactive nitrogen species

ABSTRACT Macrophages produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to bacterial infections. Mycobacteria are relatively resistant to ROS, but RNS inhibit growth of, and possibly even kill, mycobacteria in activated macrophages. We recently constructed a Mycobacterium marinum mel2 locus mutant, which is known to affect macrophage infection. We found previously that the mel2 locus confers resistance to ROS and RNS in laboratory medium, suggesting that this locus might play a similar role during growth in macrophages. Since J774A.1 murine macrophages produce high levels of ROS and RNS upon activation with gamma interferon (IFN-γ), we examined the effects of IFN-γ on ROS and RNS production by these cells as well as the effects on growth of M. marinum in these cells. We found that an M. marinum mutant with mutation of the first gene in the mel2 locus, melF, is defective for growth in IFN-γ-plus-lipopolysaccharide-treated J774A.1 cells and that this defect is abrogated by the presence of either inhibitors of nitric oxide synthase or ROS scavengers. Furthermore, the M. marinum melF mutant displays a defect at late stages in the mouse footpad model of infection. These phenotypic characteristics could be complemented fully by the entire mel2 locus but only partially by the presence of melF alone, supporting data suggesting that this insertion mutation has polar effects on downstream genes in the mel2 locus. These observations demonstrate that the M. marinum mel2 locus plays a role in resistance to ROS and RNS produced by activated macrophages.

The Mycobacterium marinum mel2 locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species.

Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood. To better understand the role of the M. marinum mel2 locus, we examined these genes for conserved motifs in silico. Striking similarities were observed between the mel2 locus and loci that encode bioluminescence in other bacterial species. Since bioluminescence systems can play a role in resistance to oxidative stress, we postulated that the mel2 locus might be important for mycobacterial resistance to ROS and RNS. We found that an M. marinum mutant in the first gene in this putative operon, melF, confers increased susceptibility to both ROS and RNS. This mutant is more susceptible to ROS and RNS together than either reactive species alone. These observations support a role for the M. marinum mel2 locus in resistance to oxidative stress and provide additional evidence that bioluminescence systems may have evolved from oxidative defense mechanisms.