Important Virulence Mechanism Research Articles

Hijacking of Host Plasminogen by Mesomycoplasma (Mycoplasma) hyopneumoniae via GAPDH: an Important Virulence Mechanism To Promote Adhesion and Extracellular Matrix Degradation.

Mesomycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world's swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process of M. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain of M. hyopneumoniae than in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface of M. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the tissue ECM barrier. IMPORTANCE Mesomycoplasma hyopneumoniae is a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants of M. hyopneumoniae are not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor in M. hyopneumoniae that facilitates the dissemination of M. hyopneumoniae by hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines against M. hyopneumoniae.

Platelet Activation and Aggregation Induced by Streptococcus bovis/Streptococcus equinus Complex.

Streptococcus bovis/Streptococcus equinus complex (SBSEC) is a common cause of infective endocarditis (IE). For IE-pathogens, the capacity to activate and aggregate platelets is believed to be an important virulence mechanism. While the interactions between bacteria and platelets have been described in detail for many Gram-positive pathogens, little research has been carried out with SBSEC in this respect. Twenty-six isolates of the four most common species and subspecies of SBSEC identified in bacteremia were collected, and interactions with platelets were investigated in platelet rich plasma (PRP) from three donors. Aggregation was studied using light-transmission aggregometry and platelet activation using flow cytometry detecting surface upregulation of CD62P. Platelets and serum were treated with different inhibitors to determine mechanisms involved in platelet aggregation and activation. Twenty-two of 26 isolates induced aggregation in at least one donor, and four isolates induced aggregation in all three donors. In PRP from donor 1, isolate SL1 induced a rapid aggregation with a median time of 70 s to reach 50% aggregation. Blockade of the platelet Fc-receptor or enzymatic cleavage of IgG abolished platelet activation and aggregation. The capacity for bacteria-induced platelet aggregation was also shown to be transferable between donors through serum. SBSEC mediates platelet aggregation in an IgG and IgG-Fc-receptor dependent manner. Bacterial activation of platelets through this pathway is common for many bacteria causing IE and could be a potential therapeutic target for the prevention and treatment of this infection. IMPORTANCE The capacity of bacteria to activate and aggregate platelets is believed to contribute to the pathogenesis of IE. The Streptococcus bovis/Streptococcus equinus complex (SBSEC) contains known IE-pathogens, but there is limited research on the different subspecies ability to interact with platelets and what signaling pathways are involved. This study reports that 22 of 26 tested isolates of different subspecies within SBSEC can induce aggregation, and that aggregation is host dependent. The Fc-IgG-receptor pathway was shown essential for platelet activation and aggregation. To the best of our knowledge, this is the first study that reports on platelet interactions of SBSEC-isolates other than Streptococcus gallolyticus subspecies gallolyticus as well as the first study to report of mechanisms of platelet interaction of SBSEC-isolates. It adds SBSEC to a group of bacteria that activate and aggregate platelets via the platelet Fc-receptor. This could be a potential therapeutic target for prevention of IE.

Characterization of Staphylococcus aureus biofilms via crystal violet binding and biochemical composition assays of isolates from hospitals, raw meat, and biofilm-associated gene mutants

Staphylococcus aureus (SA) is a gram-positive coccus and an opportunistic pathogen of humans. The ability of SA to form biofilms is an important virulence mechanism because biofilms are protected from host immune responses and antibiotic treatment. This study examines the relative biofilm strength of a variety of hospital and meat-associated strains of SA, using a crystal violet (CV) staining assay. Biofilms were treated with either DNase or proteinase K prior to CV staining, and compared to mock-treated results, to better understand the biochemical composition. Biofilm polysaccharide concentration was also measured using the phenol sulfuric-acid assay which was normalized to base biofilm strength. We found that hospital-associated isolates have biofilms that bind significantly more CV than for meat isolates and are significantly more protein and polysaccharide-based while meat isolates have significantly more DNA-based biofilms. This study also investigates the effects that biofilm-related genes have on biofilm formation and composition by analyzing specific transposon mutants of genes previously shown to play a role in biofilm development. agrA, atl, clfA, fnbA, purH, and sarA mutants produce significantly weaker biofilms (bind less CV) as compared to a wild-type control, whereas the acnA mutant produces a significantly stronger biofilm. Biofilms formed from these mutant strains were treated (or mock-treated) with DNase or proteinase K and tested with phenol and sulfuric acid to determine what role these genes play in biofilm composition. The acnA, clfA, fnbA, and purH mutants showed significant reduction in biofilm staining after either proteinase K or DNase treatment, agrA and sarA mutants showed significant biofilm reduction after only proteinase K treatment, and an atl mutant did not show significant biofilm reduction after either proteinase K or DNase treatment. These data suggest that biofilms that form without acnA, clfA, fnbA, and purH are DNA- and protein-based, that biofilms lacking agrA and sarA are mainly protein-based, and biofilms lacking atl are mainly polysaccharide-based. These results help to elucidate how these genes affect biofilm formation and demonstrate how mutating biofilm-related genes in SA can cause a change in biofilm composition.

Cryptococcus spp. and Cryptococcosis: focusing on the infection in Brazil.

Cryptococcosis is a global fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii yeast complex. This infection is acquired by inhalation of propagules such as basidiospores or dry yeast, initially causing lung infections with the possibility of progressing to the meninges. This infection mainly affects immunocompromised HIV and transplant patients; however, immunocompetent patients can also be affected. This review proposes to evaluate cryptococcosis focusing on studies of this mycosis in Brazilian territory; moreover, recent advances in the understanding of its virulence mechanism, animal models in research are also assessed. For this, literature review as realized in PubMed, Scielo, and Brazilian legislation. In Brazil, cryptococcosis has been identified as one of the most lethal fungal infections among HIV patients and C. neoformans VNI and C. gattii VGII are the most prevalent genotypes. Moreover, different clinical settings published in Brazil were described. As in other countries, cryptococcosis is difficult to treat due to a limited therapeutic arsenal, which is highly toxic and costly. The presence of a polysaccharide capsule, thermo-tolerance, production of melanin, biofilm formation, mechanisms for iron use, and morphological alterations is an important virulence mechanism of these yeasts. The introduction of cryptococcosis as a compulsory notification disease could improve data regarding incidence and help in the management of these infections.

Inhibition of Campylobacter jejuni Biofilm Formation by D-Amino Acids.

The ability of bacterial pathogens to form biofilms is an important virulence mechanism in relation to their pathogenesis and transmission. Biofilms play a crucial role in survival in unfavorable environmental conditions, acting as reservoirs of microbial contamination and antibiotic resistance. For intestinal pathogen Campylobacter jejuni, biofilms are considered to be a contributing factor in transmission through the food chain and currently, there are no known methods for intervention. Here, we present an unconventional approach to reducing biofilm formation by C. jejuni by the application of D-amino acids (DAs), and L-amino acids (LAs). We found that DAs and not LAs, except L-alanine, reduced biofilm formation by up to 70%. The treatment of C. jejuni cells with DAs changed the biofilm architecture and reduced the appearance of amyloid-like fibrils. In addition, a mixture of DAs enhanced antimicrobial efficacy of D-Cycloserine (DCS) up to 32% as compared with DCS treatment alone. Unexpectedly, D-alanine was able to reverse the inhibitory effect of other DAs as well as that of DCS. Furthermore, L-alanine and D-tryptophan decreased transcript levels of peptidoglycan biosynthesis enzymes alanine racemase (alr) and D-alanine-D-alanine ligase (ddlA) while D-serine was only able to decrease the transcript levels of alr. Our findings suggest that a combination of DAs could reduce biofilm formation, viability and persistence of C. jejuni through dysregulation of alr and ddlA.

Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B.

Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 μg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

Human IgM Inhibits the Formation of Titan-Like Cells in Cryptococcus neoformans.

Human studies have shown associations between cryptococcal meningitis and reduced IgM memory B cell levels, and studies in IgM- and/or B cell-deficient mice have demonstrated increased Cryptococcus neoformans dissemination from lungs to brain. Since immunoglobulins are part of the immune milieu that C. neoformans confronts in a human host, and its ability to form titan cells is an important virulence mechanism, we determined the effect of human immunoglobulins on C. neoformans titan cell formation in vitro (i) Fluorescence microscopy showed normal human IgG and IgM bind C. neoformans (ii) C. neoformans grown in titan cell-inducing medium with IgM, not IgG, inhibited titan-like cell formation. (iii) Absorption of IgM with laminarin or curdlan (branched and linear 1-3-beta-d-glucans, respectively) decreased this effect. (iv) Transmission electron microscopy revealed that cells grown with IgM had small capsules and unique features not seen with cells grown with IgG. (v) Comparative transcriptional analysis of cell wall, capsule, and stress response genes showed that C. neoformans grown with IgM, not IgG or phosphate-buffered saline (PBS), had decreased expression of chitin synthetase, CHS1, CHS2, and CHS8, and genes encoding cell wall carbohydrate synthetases α-1-3-glucan (AGS1) and β-1,3-glucan (FKS1). IgM also decreased expression of RIM101 and HOG1, genes encoding central regulators of C. neoformans stress response pathways and cell morphogenesis. Our data show human IgM affects C. neoformans morphology in vitro and suggest that the hypothesis that human immunoglobulins may affect C. neoformans virulence in vivo warrants further investigation.

Broad and Effective Protection against Staphylococcus aureus Is Elicited by a Multivalent Vaccine Formulated with Novel Antigens.

The demand for a prophylactic vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has motivated numerous dedicated research groups to design and develop such a vaccine. In this study, we have developed a multivalent vaccine, Sta-V5, composed of five conserved antigens involved in three important virulence mechanisms. This prototype vaccine conferred up to 100% protection against multiple epidemiologically relevant S. aureus isolates in five different murine disease models. The vaccine not only elicits functional antibodies that mediate opsonophagocytic killing of S. aureus but also mounts robust antigen-specific T-cell responses. In addition, our data implied that γδ T cells contribute to the protection induced by Sta-V5 in a murine skin infection model.IMPORTANCEStaphylococcus aureus infections, especially MRSA infections, are becoming a major global health issue and are resulting in mortality rates that are increasing every year. However, an effective vaccine is lacking due to the complexity of the infection process of S. aureus In this study, we found that the addition of two novel protein components to three well-studied vaccine candidates significantly improved the efficacy of the combined vaccine. Furthermore, the five-component vaccine not only elicits a robust antibody response but also induces cytokine secretion by T cells, making it a promising vaccine candidate to fill the void.

Cryptococcus neoformans Escape From Dictyostelium Amoeba by Both WASH-Mediated Constitutive Exocytosis and Vomocytosis.

Cryptococcus neoformans is an environmental yeast that can cause opportunistic infections in humans. As infecting animals does not form part of its normal life-cycle, it has been proposed that the virulence traits that allow cryptococci to resist immune cells were selected through interactions with environmental phagocytes such as amoebae. Here, we investigate the interactions between C. neoformans and the social amoeba Dictyostelium discoideum. We show that like macrophages, D. discoideum is unable to kill C. neoformans upon phagocytosis. Despite this, we find that the yeast pass through the amoebae with an apparently normal phagocytic transit and are released alive by constitutive exocytosis after ~80 min. This is the canonical pathway in amoebae, used to dispose of indigestible material after nutrient extraction. Surprisingly however, we show that upon either genetic or pharmacological blockage of constitutive exocytosis, C. neoformans still escape from D. discoideum by a secondary mechanism. We demonstrate that constitutive exocytosis-independent egress is stochastic and actin-independent. This strongly resembles the non-lytic release of cryptococci by vomocytosis from macrophages, which do not perform constitutive exocytosis and normally retain phagocytosed material. Our data indicate that vomocytosis is functionally redundant for escape from amoebae, which thus may not be the primary driver for its evolutionary selection. Nonetheless, we show that vomocytosis of C. neoformans is mechanistically conserved in hosts ranging from amoebae to man, providing new avenues to understand this poorly-understood but important virulence mechanism.

Skin and Soft Tissue Infections Due to Nontuberculous Mycobacteria.

This review describes recent trends in the epidemiology of nontuberculous mycobacteria (NTM), emerging pathogens, new insights into NTM pathogenesis, and advances in diagnosis and treatment. Emerging pathogens include Mycobacterium chimaera and drug-resistant subspecies of Mycobacterium abscessus. Important virulence mechanisms of pathogenic NTM include the ability to alter the macrophage's permissiveness to intracellular bacterial growth. New diagnostic tools consist of DNA probes, gene sequencing, and matrix-assisted laser desorption ionization-time of flight. These methods allow rapid speciation of NTM species, in some cases directly from patient samples. There are few novel agents available to treat NTM, although some repurposed drugs show excellent activity. The incidence of NTM infections appears to be increasing in a number of regions around the world. Molecular methods are now the diagnostic tools of choice. Discovery of novel effective agents and/or drug combinations with greater likelihood of cure, shorter treatment duration, and fewer side effects are research priorities.

The Overlooked Glycan Components of the Cryptococcus Capsule.

Pathogenic species of Cryptococcus kill approximately 200,000 people each year. The most important virulence mechanism of C. neoformans and C. gattii, the causative agents of human and animal cryptococcosis, is the ability to form a polysaccharide capsule. Acapsular mutants of C. neoformans are avirulent in mice models of infection, and extracellularly released capsular polysaccharides are deleterious to the immune system. The principal capsular component in the Cryptococcus genus is a complex mannan substituted with xylosyl and glucuronyl units, namely glucuronoxylomannan (GXM). The second most abundant component of the cryptococcal capsule is a galactan with multiple glucuronyl, xylosyl, and mannosyl substitutions, namely glucuronoxylomannogalactan (GXMGal). The literature about the structure and functions of these two polysaccharides is rich, and a number of comprehensive reviews on this topic are available. Here, we focus our discussion on the less explored glycan components associated with the cryptococcal capsule, including mannoproteins and chitin-derived molecules. These glycans were selected for discussion on the basis that i) they have been consistently detected not only in the cell wall but also within the cryptococcal capsular network and ii) they have functions that impact immunological and/or pathogenic mechanisms in the Cryptococcus genus. The reported functions of these molecules strongly indicate that the biological roles of the cryptococcal capsule go far beyond the well-known properties of GXM and GXMGal.

Leptospira Infection Interferes with the Prothrombinase Complex Assembly during Experimental Leptospirosis

Leptospirosis is a worldwide zoonotic and neglected infectious disease of human and veterinary concern, caused by pathogenic Leptospira species. Although bleeding is a common symptom of severe leptospirosis, the cause of hemorrhage is not completely understood. In severe infections, modulation of hemostasis by pathogens is an important virulence mechanism, and hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. Here, we analyze the coagulation status of experimentally infected hamsters in an attempt to determine coagulation interferences and the origin of leptospirosis hemorrhagic symptomatology. Hamsters were experimentally infected with L. interrogans. The lungs, kidneys, and livers were collected for culture, histopathology, and coagulation assays. L. interrogans infection disturbs normal coagulation in the organs of animals. Our results suggest the presence of a thrombin-like factor or FX activator, which is able to activate FII in the leptospirosis organ extracts. The activity of those factors is accelerated in the prothrombinase complex. Additionally, we show for the first time that live leptospires act as a surface for the prothrombinase complex assembly. Our results contribute to the understanding of leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments in the severe manifestations of the disease.

Real-time measurement of Plasmodium falciparum-infected erythrocyte cytoadhesion with a quartz crystal microbalance.

BackgroundAn important virulence mechanism of the malaria parasite Plasmodium falciparum is cytoadhesion, the binding of infected erythrocytes to endothelial cells in the second half of asexual blood stage development. Conventional methods to investigate adhesion of infected erythrocytes are mostly performed under static conditions, many are based on manual or semi-automated read-outs and are, therefore, difficult to standardize. Quartz crystal microbalances (QCM) are sensitive to nanogram-scale changes in mass and biomechanical properties and are increasingly used in biomedical research. Here, the ability of QCM is explored to measure binding of P. falciparum-infected erythrocytes to two receptors: CD36 and chondroitin sulfate A (CSA) under flow conditions.MethodsBinding of late stage P. falciparum parasites is measured in comparison to uninfected erythrocytes to CD36- and CSA-coated quartzes by QCM observing frequency shifts. CD36-expressing cell membrane fragments and CSA polysaccharide were coated via poly-l-lysine to the quartz. The method was validated by microscopic counting of attached parasites and of erythrocytes to the coated quartzes.ResultsFrequency shifts indicating binding of infected erythrocytes could be observed for both receptors CD36 and CSA. The frequency shifts seen for infected and uninfected erythrocytes were strongly correlated to the microscopically counted numbers of attached cells.ConclusionsIn this proof-of-concept experiment it is shown that QCM is a promising tool to measure binding kinetics and specificity of ligand-receptor interactions using viable, parasite-infected erythrocytes. The method can improve the understanding of the virulence of P. falciparum and might be used to cross-validate other methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1374-7) contains supplementary material, which is available to authorized users.

Experimental Intrastromal Antibiotic Injection for Refractory Keratits Caused by Biofilm Forming Pseudomonas Aeruginosa

Background: Refractory keratitis is a major ophthalmic concern, unresponsive patients to ordinary or fortified eye drops or even specific antibiotic drops according to the cultures taken from the lesions may necessitate therapeutic keratoplasty with poor prognosis. Biofilm is an important virulence mechanism for bacterial infections and is a major cause of antibiotic resistance. Treatment of biofilm requires delivering of higher antibiotic concentration than normal. Objectives: This study aims to evaluate the role of biofilm in formation of keratitis and to consider intrastromal antibiotic injection to deliver higher concentration of antibiotics to treat keratitis caused by biofilm forming pseudomonas. Methodology: We used 40 New Zealand white female rabbits in this prospective in-vivo experimental study. Rabbits were classified in to four groups: one eye (the right one) in all rabbits was used for the inoculation with the organism as a test eye while the left eye was used as a control. The right eye of group (1) was inoculated with biofilm forming Pseudomonas aeruginosa and treated with intrastromal ciprofloxacin injection, while, first eye of group (2) was inoculated with biofilm forming Pseudomonas aeruginosa and treated with ciprofloxacin eye drops, Meanwhile, the first eye of group (3) was inoculated with non- biofilm forming Pseudomonas aeruginosa and treated with intrastromal ciprofloxacin injection, At last, the first eye of the group (4) was inoculated with biofilm forming Pseudomonas aeruginosa and treated with ciprofloxacin eye drops, the second eye was always the control eye. Results: Using ANOVA and LSD tests to compare the means of ulcer diameters in different groups, we found that biofiom is a virulence factor that plays an important role keratitis and there is a great reduction in ulcer size in group receiving intrastromal antibiotic injection compared with the those received eye drops. Conclusion: Biofilm is an important virulence factor that have to be investigated during the diagnostic course of keratitis and intrastromal ciprofloxacin injection is an effective mean of treating keratitis that can provide an alternative way for surgery to treat refractory keratitis.

What Can Proteomics Tell Us about Tuberculosis?

Tuberculosis (TB) is an infectious disease transmitted by aerosol droplets and characterized by forming granulomatous lesions. Although the number of people infected in the population is high, the vast majority does not exhibit symptoms of active disease and only 5-10% develop the disease after a latent period that can vary from weeks to years. The bases of the immune response for this resistance are unknown, but it depends on a complex interaction between the environment, the agent, and the host. The analysis of cellular components of M. tuberculosis shows important host-pathogen interactions, metabolic pathways, virulence mechanisms, and mechanisms of adaptation to the environment. However, the M. tuberculosis proteome still remains largely uncharacterized in terms of virulence and pathogenesis. Here, we summarize some of the major proteomic studies performed to scrutinize all the mycobacterial components.

Pantoea ananatis Utilizes a Type VI Secretion System for Pathogenesis and Bacterial Competition.

Type VI secretion systems (T6SSs) are a class of macromolecular machines that are recognized as an important virulence mechanism in several gram-negative bacteria. The genome of Pantoea ananatis LMG 2665(T), a pathogen of pineapple fruit and onion plants, carries two gene clusters whose predicted products have homology with T6SS-associated gene products from other bacteria. Nothing is known regarding the role of these T6SS-1 and T6SS-3 gene clusters in the biology of P. ananatis. Here, we present evidence that T6SS-1 plays an important role in the pathogenicity of P. ananatis LMG 2665(T) in onion plants, while a strain lacking T6SS-3 remains as pathogenic as the wild-type strain. We also investigated the role of the T6SS-1 system in bacterial competition, the results of which indicated that several bacteria compete less efficiently against wild-type LMG 2665(T) than a strain lacking T6SS-1. Additionally, we demonstrated that these phenotypes of strain LMG 2665(T) were reliant on the core T6SS products TssA and TssD (Hcp), thus indicating that the T6SS-1 gene cluster encodes a functioning T6SS. Collectively, our data provide the first evidence demonstrating that the T6SS-1 system is a virulence determinant of P. ananatis LMG 2665(T) and plays a role in bacterial competition.

The flavonoid (-)-epicatechin affects cytoskeleton proteins and functions in Entamoeba histolytica.

This work contributes with some advances in the action mechanisms involved in the antiamoebic effect of the flavonoid (-)-epicatechin. We found that this flavonoid has an unusual effect on trophozoites growth that is dependent of its concentration. Additionally, we reported that (-)-epicatechin affects mainly amebic cytoskeleton proteins, which results in alteration on important virulence mechanisms, like adhesion, migration, phagocytosis and cytolysis. This study provides new knowledge about a potential alternative therapy directed to the treatment of amoebiasis.

Screening for inhibition of Vibrio cholerae VipA-VipB interaction identifies small-molecule compounds active against type VI secretion.

The type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB in Vibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an α-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth of Vibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A1 activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted by in silico analysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance.

An in silico study on molecular level interactions of host Siderocalin with siderophores from Mycobacterium tuberculosis and other bacterial species

Background Tuberculosis (TB) drug research and development has witnessed resurgence in recent times primarily due to the high mortality rates despite the availability of front line drugs and a prominent BCG vaccine. Synergy of TB with HIV is another factor which has warranted a search for newer therapeutic interventions. Treatment of latent TB infection is also the need of the hour. Iron acquisition is an important virulence mechanism of Mycobacterium tuberculosis (MTB). To scavenge iron, bacterial species have developed high affinity, low molecular weight iron chelators termed as siderophores. To counteract the detrimental effect of microbial iron acquisition systems, the host secretes a 21kDa lipocalin protein (Siderocalin) that binds with these iron laden siderophores in an attempt to restrict the growth of MTB within host macrophages.

Mutations in the Pseudomonas aeruginosa Needle Protein Gene pscF Confer Resistance to Phenoxyacetamide Inhibitors of the Type III Secretion System

The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics in P. aeruginosa infections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition of P. aeruginosa T3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS gene pscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles of pscF, together with its chaperone and cochaperone genes pscE and pscG, to a ΔpscF P. aeruginosa strain demonstrated that each of the single-codon mutations in pscF is necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.