Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonize the host at the cellular level. These factors are either secretory, membrane associated or cytosolic in nature. The cytosolic factors facilitate the bacterium to undergo quick adaptive—metabolic, physiological and morphological shifts. The membrane associated virulence factors aid the bacterium in adhesion and evasion of the host cell. The secretory factors are important components of bacterial armoury which help the bacterium wade through the innate and adaptive immune response mounted within the host. In extracellular pathogens, the secretory virulence factors act synergistically to kill the host cells. In this review, we revisit the role of some of the secreted virulence factors of two human pathogens: Mycobacterium tuberculosis—an intracellular pathogen and Bacillus anthracis—an extracellular pathogen. The advances in research on the role of secretory factors of these pathogens during infection are discussed.

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Bacterial Virulence Factors: Secreted for Survival

Functional relationships between proteins that do not share global structure similarity can be established by detecting their ligand-binding-site similarity. For a large-scale comparison, it is critical to accurately and efficiently assess the statistical significance of this similarity. Here, we report an efficient statistical model that supports local sequence order independent ligand–binding-site similarity searching. Most existing statistical models only take into account the matching vertices between two sites that are defined by a fixed number of points. In reality, the boundary of the binding site is not known or is dependent on the bound ligand making these approaches limited. To address these shortcomings and to perform binding-site mapping on a genome-wide scale, we developed a sequence-order independent profile–profile alignment (SOIPPA) algorithm that is able to detect local similarity between unknown binding sites a priori. The SOIPPA scoring integrates geometric, evolutionary and physical information into a unified framework. However, this imposes a significant challenge in assessing the statistical significance of the similarity because the conventional probability model that is based on fixed-point matching cannot be applied. Here we find that scores for binding-site matching by SOIPPA follow an extreme value distribution (EVD). Benchmark studies show that the EVD model performs at least two-orders faster and is more accurate than the non-parametric statistical method in the previous SOIPPA version. Efficient statistical analysis makes it possible to apply SOIPPA to genome-based drug discovery. Consequently, we have applied the approach to the structural genome of Mycobacterium tuberculosis to construct a protein–ligand interaction network. The network reveals highly connected proteins, which represent suitable targets for promiscuous drugs.

Contact: [email protected]

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A unified statistical model to support local sequence order independent similarity searching for ligand-binding sites and its application to genome-based drug discovery

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.

In vivo induced antigen technology (IVIAT).

Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress

Uncomplicated urinary tract infections (UTI) are caused most commonly by uropathogenic Escherichia coli (UPEC). Whole-genome screening approaches, including transcriptomic, proteomic, and signature-tagged mutagenesis, have shown that UPEC highly expresses or requires genes for translational machinery, capsule, lipopolysaccharide, type 1 fimbriae, and iron acquisition systems during UTI. To identify additional genes expressed by UPEC during UTI, an immunoscreening approach termed in vivo-induced antigen technology (IVIAT) was employed to identify antigens produced during experimental infection that are not produced during in vitro culture. An inducible protein expression library, constructed from genomic DNA isolated from UPEC strain CFT073, was screened using exhaustively adsorbed pooled sera from 20 chronically infected female CBA/J mice. Using this approach, we identified 93 antigens induced by UPEC in vivo. A representative subset of these genes was tested by quantitative PCR for expression by CFT073 in vivo and during growth in human urine or LB medium in vitro; proWX, narJI, lolA, lolD, tosA (upxA), c2432, katG, ydhX, kpsS, and yddQ were poorly expressed in vitro but highly expressed in vivo. Of these, tosA, a gene encoding a predicted repeat-in-toxin family member, was expressed exclusively during UTI. Deletion of tosA in UPEC strain CFT073 resulted in significant attenuation in bladder and kidney infections during ascending UTI. By screening for in vivo-induced antigens, we identified a novel UPEC virulence factor and additional proteins that could be useful as potential vaccine targets.

Identification of in vivo-induced antigens including an RTX family exoprotein required for uropathogenic Escherichia coli virulence.

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach

Mycobacterium tuberculosis codes for a HAD-phosphatase, Rv3042c (MtSerB2), that has earlier been characterized as a metabolic enzyme. Here we demonstrate that MtSerB2 is secreted into the cytosol of infected macrophages and is found in bronchoalveolar lavage samples of tuberculosis patients. MtSerB2 induces significant cytoskeleton rearrangements through cofilin activation and affects the expression of genes that regulate actin dynamics. It specifically interacts with HSP90, HSP70 and HSP27 that block apoptotic pathways and not with other HSPs. It actively dephosphorylates MAPK-p38 and NF-kappa B p65 that play crucial roles in inflammatory and immune responses. This in turn leads to down-regulation of Interleukin 8, a chemotactic and inflammatory cytokine. Finally, during evaluation of inhibitors against MtSerB2 we found that Clofazimine, a drug being evaluated for XDR and MDR tuberculosis, inhibits MtSerB2 phosphatase activity and reverses the above effects and interactions with host proteins. Overall, the study identifies that MtSerB2 has new functions that might help the pathogen to evade the host’s immune response.

The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine.

Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem because treatment is complicated, cure rates are well below those for drug susceptible tuberculosis (TB), and patients may remain infectious for months or years despite receiving the best available therapy To gain a better understanding of MDR-TB, we characterized isolates recovered from 69 patients with MDR-TB, by use of IS6110 restriction fragment-length polymorphism (RFLP) analysis; spacer oligonucleotide genotyping (ie spoligotyping) Clinical isolates from patients with tuberculosis have been considered to contain clonally expanded Mycobacterium tuberculosis (MTB) strain Over the years, the identification method based on IS6110 insertion sequences has been established as the standard for typing strains of MTB IS6110 RFLP fingerprinting is very convincing when it is applied to classify MTB isolates harboring a large number of IS6110 in their chromosomes Therefore, in the present study we have characterized the isolates from the patients suffering from MDR TB, on the basis of conserved Variable Number Tandem Repeats (VNTR), Direct Repeats (DR) and Insertion Sequences (IS) IS6110 elements The polymorphic data showed significant level of dissimilarities among all the MDR isolates of MTB Comparative studies with the DR and VNTR data substantiate that polymorphism occur among MDR-TB cases as shown by the number of repeats present in different clinical isolates

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Kanchan Srivastava

Papers

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach

The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine.

Molecular Profiling of Drug Resistant Isolates of Mycobacterium tuberculosis in North India