Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene

The virulence of the mycobacteria that cause tuberculosis depends on their ability to multiply in mammalian hosts. Disruption of the bacterial erp gene, which encodes the exported repetitive protein, impaired multiplication of M. tuberculosis and M. bovis Bacille Calmette-Guérin in cultured macrophages and mice. Reintroduction of erp into the mutants restored their ability to multiply. These results indicate that erp contributes to the virulence of M. tuberculosis.     

Effects of overexpression of the alkyl hydroperoxide reductase AhpC on the virulence and isoniazid resistance of Mycobacterium tuberculosis

Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.     

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.     

Growth of virulent and avirulent Mycobacterium tuberculosis strains in human macrophages

Mycobacterium tuberculosis H37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth of M. tuberculosis strains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics of M. tuberculosis strains in animal models are relevant to human tuberculosis infection.     

Identification and subcellular localization of a novel Cu,Zn superoxide dismutase of Mycobacterium tuberculosis

Periplasmic copper, zinc superoxide dismutases (Cu,ZnSOD) of several Gram-negative pathogens have been shown to play an important role in protection against exogenous superoxide radicals and in determining virulence of the pathogens. Here we report the cloning and characterization of the sodC gene, encoding Cu,ZnSOD, from the Gram-positive Mycobacterium tuberculosis. The predicted protein sequence contains 240 amino acids with a putative signal peptide at the N-terminus and shows approximately 25% identity to other bacterial sodC. Recombinant proteins of a full-length sodC and a truncated form lacking the putative signal peptide were overexpressed in Escherichia coli and affinity purified. Renatured recombinant M. tuberculosis sodC protein possessed characteristics of a Cu,ZnSOD. Immunoblotting with an antiserum against the recombinant M. tuberculosis Cu,ZnSOD allowed detection of a single polypeptide in the lysate of M. tuberculosis. This polypeptide has a similar size as the recombinant protein without the putative signal peptide indicating that the endogenous Cu,ZnSOD in M. tuberculosis might be processed and secreted. Furthermore, immunogold electron microscopic image showed that Cu,ZnSOD is located in the periphery of M. tuberculosis. The enzymatic activity and subcellular localization of this novel Cu,ZnSOD suggest that it may play a role in determining virulence of M. tuberculosis.     

The microbiological diagnosis of tuberculosis in areas under strict radiation control

Mycobacterial variability was studied in active tuberculosis patients from radioactive contamination area subject to close radiation control. For such areas it was noted that the patients discharge altered coccoid mycobacteria stained with routine luminescent dyes. Biological investigations of freshly isolated strains revealed potentiation of M. tuberculosis virulence in patients with respiratory tuberculosis. A large number of patients from the contaminated areas against control demonstrated primary resistance to rifampicin and isoniazid.     

Characteristics and outcome of anti-glomerular basement membrane disease: a single-center experience

The relative virulence and avirulence of Mycobacterium tuberculosis strains H37Rv and H37Ra were previously defined using animal infection models. To investigate host species' specificity of mycobacterial virulence, growth of the 2 M. tuberculosis strains in human monocyte-derived macrophages in vitro was studied. Mycobacterial growth was evaluated by acid-fast staining, electron microscopy, and colony-forming units (cfu) assay. As expected, the 2 strains demonstrated significantly different growth rates in mouse macrophages in vitro (53 h for H37Rv, 370 h for H37Ra). In marked contrast, in human macrophages the average division times of the strains were nearly equal (80 h for H37Rv and 76 h for H37Ra by cfu measurement, and 96 h for H37Rv and 104 h for H37Ra by acid-fast staining). These findings indicate that observations of mycobacterial virulence in murine systems may not necessarily translate to the human system, in which different mechanisms to control mycobacterial growth may be expressed.     

Drug-resistant strains of Mycobacterium tuberculosis exhibit a range of virulence for mice

A panel of clinical isolates of Mycobacterium tuberculosis, several of which were resistant to one or more antimycobacterial drugs, were tested for their capacity to give rise to active disease following aerogenic infection of normal immunocompetent mice. The panel exhibited a range of virulence in this model, which followed no clear trend in terms of geographical source, degree of drug resistance, or rate of growth in vitro. Several isolates grew very quickly over the first 20 days in mouse lungs before being contained by emerging immunity. In view of this latter observation, we hypothesize that it is possible that such so-called fast growers may be responsible for the rapid fatality sometimes seen in immunocompromised patients with tuberculosis. Moreover, the results of the study do not support the belief that increased drug resistance usually associates with loss of virulence of the isolate.     

Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin

BACKGROUND: Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron. Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion. The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively. RESULTS: A ten-gene cluster spanning 24 kilobases of the M. tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis. The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, MbtI an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase. An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation. Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP. CONCLUSIONS: Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.     

BCG vaccination

The author expounds the fairly obscure history of BCG vaccination. Summarises the efficiency of accomplished vaccinations of different geographical areas. He accounts the diversity of results by the distinction of technical and methodical conditions and disparity in virulence of BCG strains. Overviews the Inland and European routine and the risk of possible complications. The favourable position of domestic childhood tuberculosis is due to the effective preventive arrangements. Considering the international and national epidemiology of tuberculosis in adults the administration of BCG vaccine, by chance in modified form, is recommended further on.     

Large-scale identification of virulence genes from Streptococcus pneumoniae

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.     

Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.     

New information on the structure of mycobacteria

Recent progress about the mycobacterial structures have been realized and two major structures have been concerned: the genome and the cell wall. From these acquired new knowledge several lines of clinical research and diagnosis application emerged. Cloning and sequencing of several mycobacterial genes led to the development of diagnostic tools (DNA probes, PCR, finger printings of indated mycobacterial strains) and the potential detection of multiding resistant strains of M. tuberculosis. Genetic manipulations involving various mycobacterial genes do open the way for more precise molecular approaches concerning virulence factors involved in the pathophysiological understanding of mycobacterial diseases. Comparative physico-chemical and ultra-structural analysis of the mycobacterial cell wall evoked a highly complexed cell wall structure, constituted of a double lipidic layer linked to the peptidoglycan (PG). The first layer is constituted of mycolic acids that are linked to the PG by arabinogalactan, and to the superficial layer by hydrophobic interactions of glycolipids. The superficial layer is constituted of amphiphatic glycolipids, having a lipidic banal pole and a polysaccharidic apical pole. The knowledge of the mycobacterial cell wall structure opened the way of: the development of immunological diagnostic tools, being now days in clinical evaluation phase, a better approach for host-bacteria relationship study at the cellular level (macrophage, lymphocytes), and the understanding of the mode of action of antimycobacterial drugs such as isoniazid and ethambutol.     

Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis

To investigate the role of chemokines during the initial local response to Mycobacterium tuberculosis in the human lung, we studied chemokine production by the human alveolar epithelial cell line A549 after infection with M. tuberculosis. M. tuberculosis-infected A549 cells produced mRNAs and protein for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) but not mRNAs for macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES. Chemokine production in response to M. tuberculosis was not dependent on production of tumor necrosis factor alpha, IL-1beta, or IL-6. Two virulent clinical M. tuberculosis isolates, the virulent laboratory strain H37Rv, and the avirulent strain H37Ra elicited production of comparable concentrations of MCP-1 and IL-8, whereas killed M. tuberculosis and three Mycobacterium avium strains did not. The three virulent M. tuberculosis strains grew more rapidly than the avirulent M. tuberculosis strain in the alveolar epithelial cell line, and the three M. avium strains did not grow intracellularly. These findings suggest that intracellular growth is necessary for mycobacteria to elicit production of MCP-1 and IL-8 by alveolar epithelial cells but that virulence and the rate of intracellular growth do not correlate with chemokine production. Alveolar epithelial cells may contribute to the local inflammatory response in human tuberculosis by producing chemokines which attract monocytes, lymphocytes, and polymorphonuclear cells.     

Bacterial N-acyl-homoserine-lactone-dependent signalling and its potential biotechnological applications

N-acyl homoserine lactones are bacterial signalling molecules involved in regulating diverse metabolic functions, particularly those relating to virulence, in concert with cell density. Each aspect of the signalling pathway, from production and recognition of the signal to expression of the target genes, offers a potential opportunity for exploitation. Attention is now focusing on the development of novel methods for bacterial enumeration, modulation of bacterial virulence and flexible, coordinated expression of heterologous genes through the use of N-acyl-homoserine-lactone-based systems.     

Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies

The ura-blaster technique for the disruption of Candida albicans genes has been employed in a number of studies to identify possible genes encoding virulence factors of this fungal pathogen. In this study, the URA3-encoded orotidine 5'-monophosphate (OMP) decarboxylase enzyme activities of C. albicans strains with ura-blaster-mediated genetic disruptions were measured. All strains harboring genetic lesions via the ura-blaster construct showed reduced OMP decarboxylase activities compared to that of the wild type when assayed. The activity levels in different gene disruptions varied, suggesting a positional effect on the level of gene expression. Because the URA3 gene of C. albicans has previously been identified as a virulence factor for this microorganism, our results suggest that decreased virulence observed in strains constructed with the ura-blaster cassette cannot accurately be attributed, in all cases, to the targeted genetic disruption. Although revised methods for validating a URA3-disrupted gene as a target for antifungal drug development could be devised, it is clearly desirable to replace URA3 with a different selectable marker that does not influence virulence.     

Candida albicans secreted aspartyl proteinases

Evidence suggests that infections with the opportunistic yeast Candida albicans are caused by several factors. Among these virulence attributes, secreted aspartyl proteinases (Saps) are widely believed to play a role during pathogenesis. Sap isoenzymes are encoded by at least eight closely related SAP genes. Antigen-antibody studies provided evidence that Sap isoenzymes are expressed in vivo and experimental infections with proteinase deficient mutants suggested a role for Saps in the virulence of C. albicans. However, only one gene product, Sap2, has been characterized in detail. In vitro studies with purified Sap(2) suggested several possible host targets but the role of each Sap isoenzyme remains unclear. The expression pattern of SAP genes proposed that Sap isoenzymes are secreted simultaneously with morphological changes such as the yeast to hyphal transition or during phenotypic switching. In addition, extracellular proteolytic activity may affect adhesion to host cells and thus may help the fungus to persist on host surfaces and to penetrate into deeper tissue. This review will deal with secretory proteinases from C. albicans as putative virulence factors and will focus on the more recent molecular aspects of the proteinases and their genes. Insights into the genetic organization and regulation of the secreted proteinases suggest not only that these enzymes may act as virulence factors of C. albicans, but that the pathogenesis of this fungus is indeed complex and multifactorial.     

Pathogenic adaptation of Escherichia coli by natural variation of the FimH adhesin

Conventional wisdom regarding mechanisms of bacterial pathogenesis holds that pathogens arise by external acquisition of distinct virulence factors, whereas determinants shared by pathogens and commensals are considered to be functionally equivalent and have been ignored as genes that could become adapted specifically for virulence. It is shown here, however, that genetic variation in an originally commensal trait, the FimH lectin of type 1 fimbriae, can change the tropism of Escherichia coli, shifting it toward a urovirulent phenotype. Random point mutations in fimH genes that increase binding of the adhesin to mono-mannose residues, structures abundant in the oligosaccharide moieties of urothelial glycoproteins, confer increased virulence in the mouse urinary tract. These mutant FimH variants, however, are characterized by increased sensitivity to soluble inhibitors bathing the oropharyngeal mucosa, the physiological portal of E. coli. This functional trade-off seems to be detrimental for the intestinal ecology of the urovirulent E. coli. Thus, bacterial virulence can be increased by random functional mutations in a commensal trait that are adaptive for a pathologic environment, even at the cost of reduced physiological fitness in the nonpathologic habitat.     

Salmonella typhimurium cob mutants are not hyper-virulent

It was previously reported that Salmonella typhimurium LT2 cob mutants defective in the biosynthesis of vitamin B12 (cobalamin) are more virulent than the wild type in mice. Here we show that the strains used previously are non-isogenic and that the proposed increase in virulence of the cob mutant strain results from an uncharacterized mutation in the "wild type" which attenuates virulence, most likely by decreasing expression of the spv genes on the virulence plasmid. As a result the cob mutant will appear as hyper-virulent. Examination of the virulence of reconstructed wild-type and cob mutant strains showed that their growth rates were similar in mice, and we conclude that vitamin B12 does not affect the virulence of S. typhimurium LT2.