Life on the inside: probing mycobacterium tuberculosis gene expression during infection

The identification of Mycobacterium tuberculosis genes specifically expressed during infection is a key step in understanding mycobacterial pathogenesis. Such genes most likely encode products required for survival within the host and for progressive infection. Recent advances in mycobacterial genetics have permitted the development of new techniques and the adaptation of existing methods to analyse mycobacterial in vivo gene expression and virulence. This has revealed a subset of M. tuberculosis genes that are differentially expressed during infection and has demonstrated that a number of components contribute to the virulence of the organism. This information is expected to provide new strategies to prevent tuberculosis infection, new targets for antimicrobial therapy and new insights into the infectious process.     

Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis

Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT-PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence-specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum-sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction - from the pneumococcal point of view - between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm-like state.     

Unravelling the mysteries of virulence gene regulation in Salmonella typhimurium

Salmonella typhimurium, which causes gastroenteritis in calves and humans as well as a typhoid-like disease in mice, uses numerous virulence factors to infect its hosts. Genes encoding these factors are regulated by many environmental conditions and regulatory pathways in vitro. Many virulence genes are specifically induced at particular sites during infection or in cultured host cells. The complex regulation of virulence genes observed in vitro may be necessary to restrict their expression to specific locations within the host. In vitro and in vivo studies provide clues about how virulence genes might be regulated in vivo. Future studies must assess the actual environmental signals and regulators that modulate each virulence gene in vivo and determine how multiple regulatory pathways are integrated to co-ordinate the appropriate expression of virulence factors at specific sites in vivo.     

In vivo induced antigen technology (IVIAT)

In vivo induced antigen technology (IVIAT) is a technique that identifies pathogen antigens that are immunogenic and expressed in vivo during human infection. IVIAT is complementary to other techniques that identify genes and their products expressed in vivo. Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during human infection, and may be appropriate for inclusion in therapeutic, vaccine or diagnostic applications.     

Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-regulation

Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including alpha hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.     

Identification of novel staphylococcal virulence genes by in vivo expression technology

We have applied in vivo expression technology (IVET) to the study of staphylococcal virulence. Using a promoter trap that relies on genetic recombination as a reporter of gene expression, we identified 45 staphylococcal genes that are induced during infection in a murine renal abscess model. Of these, only six were known previously; 11 others have homology to known non-staphylococcal genes. The known staphylococcal genes include agrA , part of a key locus regulating numerous virulence products, and a glycerol ester hydrolase, which may enhance staphylococcal survival in abscesses. We constructed 11 strains containing mutations in previously unknown ivi genes. Of these strains, seven were significantly attenuated in virulence compared with the wild-type parent. The mutagenized ivi genes may encode novel staphylococcal virulence factors.     

Inhibition of competence development, horizontal gene transfer and virulence in Streptococcus pneumoniae by a modified competence stimulating peptide

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.     

Manipulating the host to study bacterial virulence

The ability to manipulate animal hosts as well as bacterial pathogens greatly expands the utility of in vivo models of infection. For example, the construction of mice that harbor human tissues or express specific transgenes can provide ligand-receptor interactions that are essential for pathogenesis. Interactions between virulence factors and specific host defenses can sometimes be resolved by challenging selectively immuno deficient mice with bacteria containing virulence gene mutations. Transgenic animals expressing inducible reporters can be used to conveniently identify cells in which specific response pathways have been activated during infection. These and other approaches promise to improve the quality of information obtainable from in vivo assessments of pathogenesis.     

Validation of the tetracycline regulatable gene expression system for the study of the pathogenesis of infectious disease

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection. Here we have used a well-established murine model of disseminated candidiasis, which is highly dependent on both the virulence displayed by the fungal cells and on the host immune status, to validate the use of this system. We demonstrate that the pathogenesis of the wild type C. albicans CAF2-1 strain, which does not contain any tet-regulatable element, is not affected by the presence of doxycycline. Moreover levels of key cytokines, chemokines and many other biomarkers, as determined by multi-analyte profiling, remain essentially unaltered by the presence of the antibiotic during infection. Our results indicate that the levels of doxycycline needed to control the tetracycline regulatable promoter gene expression system have no detectable effect on global host responses during candidiasis. Because tet-regulatable systems are now being increasingly used in a variety of pathogenic microorganisms, these observations have wide implications in the field of infectious diseases.     

Host-induced, stage-specific virulence gene activation in Candida albicans during infection

An understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP-mediated genetic recombination. The FLP gene, encoding the site-specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P-FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid-sensitive phenotype of the colonies generated from such cells after FLP-mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.     

Engineered Salmonella allows real-time heterologous gene expression monitoring within infected zebrafish embryos

Microbial host-pathogen interactions have been traditionally well studied at genetic and physiological levels, but cell-resolution analyses have been particularly scarce. This has been especially remarkable for intracellular parasites for two major reasons: first, the inherent loss of bacteria traceability once infects its hosts; second and more important, the limited availability of genetic tools that allow a tight regulated expression of bacterial virulence genes once inside the host tissues. Here we present novel data supporting the use of zebrafish embryos to monitor Salmonella enterica serovar Thyphimurium infection. Intravenous infection of Salmonella can be easily monitored using in vivo fluorescence that allows the visualization of free-swimming bacteria through the circulatory system. Moreover, we have engineered Salmonella to voluntarily activate heterologous gene expression at any point during infection once inside the zebrafish macrophages using a salicylate-based expression system. This approach allows real-time cell-resolution in vivo monitoring of the infection. All together, this approach paves the road to cell-based resolution experiments that would be harder to mimic in other vertebrate infection models.