Members of the Mycobacterium tuberculosis group synthesize a family of long‐chain fatty acids, mycolic acids, which are located in the cell envelope. These include the non‐oxygenated α‐mycolic acid and the oxygenated keto‐ and methoxymycolic acids. The function in bacterial virulence, if any, of these various types of mycolic acids is unknown. We have constructed a mutant strain of M. tuberculosis with an inactivated hma (cmaA, mma4) gene; this mutant strain no longer synthesizes oxygenated mycolic acids, has profound alterations in its envelope permeability and is attenuated in mice.
SummaryThe antigen 85 complex of Mycobacterium tuberculosis consists of three abundantly secreted proteins. The recent characterization of a mycoloyltransferase activity associated in vitro with each of these antigens suggested that they are potentially important for the building of the unusual cell envelope of mycobacteria. To define the physiological role of these proteins, the gene coding for antigen 85C was inactivated by transposon mutagenesis. The resulting mutant was shown to transfer 40% fewer mycolates to the cell wall with no change in the types of mycolates esterifying arabinogalactan or in the composition of non-covalently linked mycolates. As a consequence, the diffusion of the hydrophobic chenodeoxycholate and the hydrophilic glycerol, but not that of isoniazid, was found to be much faster through the cell envelope of the mutant than that of the parent strain. Taken together, these data demonstrate that: (i) antigen 85C is involved directly or indirectly in the transfer of mycolates onto the cell wall of the whole bacterium; (ii) the enzyme is not specific for a given type of mycolate; and (iii) the cell wall-linked mycolate layer may represent a barrier for the diffusion of small hydrophobic and hydrophilic molecules.
Among the few characterized genes that have products involved in the pathogenicity of Mycobacterium tuberculosis, the etiological agent of tuberculosis, are those of the phthiocerol dimycocerosate (DIM) locus. Genes involved in biosynthesis of these compounds are grouped on a 50-kilobase fragment of the chromosome containing 13 genes. Analysis of mRNA produced from this 50-kilobase fragment in the wild type strain showed that this region is subdivided into three transcriptional units. Biochemical characterization of five mutants with transposon insertions in this region demonstrated that (i) the complete DIM molecules are synthesized in the cytoplasm of M. tuberculosis before being translocated into the cell wall; (ii) the genes fadD26 and fadD28 are directly involved in their biosynthesis; and (iii) both the drrC and mmpL7 genes are necessary for the proper localization of DIMs. Insertional mutants unable to synthesize or translocate DIMs exhibit higher cell wall permeability and are more sensitive to detergent than the wild type strain, indicating for the first time that, in addition to being important virulence factors, extractable lipids of M. tuberculosis play a role in the cell envelope architecture and permeability. This function may represent one of the molecular mechanisms by which DIMs are involved in the virulence of M. tuberculosis.Mycobacterium tuberculosis, the etiological agent of tuberculosis, is an intracellular pathogen that causes more human deaths than any other single infectious agent. Despite its tremendous importance as a public health problem, the molecules involved in the pathogenicity of the tubercle bacillus remain largely unknown. The mycobacterial cell envelope has long been thought to be involved in both the pathogenicity of these bacteria and their resistance to hostile environments and antibiotics. In addition to its postulated passive role through a strong resistance to degradation by host enzymes, impermeability to toxic macromolecules, and inactivation of small reactive molecules, such as reactive oxygen and nitrogen derivatives, the mycobacterial cell envelope may exert a more active role, notably by interacting with host cell receptors to facilitate uptake of the bacterium and by modulating the immune response (1).The mycobacterial envelope is unique, both in molecular composition and in the architectural arrangement of its constituents. From the cytoplasm to the external side of the bacterium, the cell envelope is composed of: (i) a plasma membrane; (ii) a cell wall consisting of a peptidoglycan covalently attached to the heteropolysaccharide arabinogalactan, which is in turn esterified by very long chain (C60 -C90) fatty acids called mycolic acids and various noncovalently attached lipids and glycolipids; and (iii) a capsule of polysaccharides, proteins, and lipids (1). In the last 50 years, considerable effort has been devoted to searching for putative virulence factors among constituents of the mycobacterial cell envelope. Two structurally related families of noncovalentl...
Summary Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild‐type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the ΔplcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT‐PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.
Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced protein secretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L. monocytogenes.
SummaryThe functions of OmpATb, the product of the ompATb gene of Mycobacterium tuberculosis and a putative porin, were investigated by studying a mutant with a targeted deletion of the gene, and by observing expression of the gene in wild-type M. tuberculosis H37Rv by real-time polymerase chain reaction (PCR) and immunoblotting. The loss of ompATb had no effect on growth under normal conditions, but caused a major reduction in ability to grow at reduced pH. The gene was substantially upregulated in wild-type bacteria exposed to these conditions. The mutant was impaired in its ability to grow in macrophages and in normal mice, although it was as virulent as the wild type in mice that lack T cells. Deletion of the ompATb gene reduced permeability to several small watersoluble substances. This was particularly evident at pH 5.5; at this pH, uptake of serine was minimal, suggesting that, at this pH, OmpATb might be the only functioning porin. These data indicate that OmpATb has two functions: as a pore-forming protein with properties of a porin, and in enabling M. tuberculosis to respond to reduced environmental pH. It is not known whether this second function is related to the porin-like activity at low pH or involves a completely separate role for OmpATB. The involvement with pH is likely to contribute to the ability of M. tuberculosis to overcome host defence mechanisms and grow in a mammalian host.
Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.
Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer.
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