Identification of distinct lymphocyte subsets responding to subcellular fractions of Mycobacterium bovis bacille calmette-Guerin (BCG)

In order to investigate the ability of Mycobacterium bovis BCG vaccination to induce immune responses toward different classes of mycobacterial antigens and the cell populations involved in such responses, proliferation of distinct human lymphocyte subsets from BCG-vaccinated donors in response to different subcellular fractions of BCG was analysed and compared with that of not sensitized subjects. Proliferation of different cell subsets was evaluated by flow cytometric determination of bromodeoxyuridine incorporated into DNA of dividing cells and simultaneous identification of cell surface markers. Although a certain degree of variability was observed among different donors, after 6 days of in vitro stimulation BCG-vaccinated subjects displayed, as a mean, a stronger blastogenic response to all the classes of antigens compared with non-sensitized ones. PPD, culture filtrates and membrane antigens induced a predominant proliferation of CD4(+) T cells. In contrast, preparations enriched in cytosolic antigens elicited strong proliferation of gamma delta(+) T cells which, as a mean, represented 55% of the proliferating cells. Although to a lesser extent, proliferation of gamma delta(+) T cells was also elicited by preparations enriched in membrane and cell wall antigens. In response to the latter preparation proliferation of CD4(+) T cells and CD16(+)/CD3(-) (natural killer (NK)) cells was observed, as well. In particular, cell wall antigens were found to induce significantly higher levels of proliferation of NK cells compared with all the other classes of antigens

In vitro bactericidal activity of human beta-defensin 3 against multidrug-resistant nosocomial strains

The antimicrobial activity of human beta-defensin 3 (hBD-3) against multidrug-resistant clinical isolates of Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii was evaluated. A fast bactericidal effect (within 20 min) against all bacterial strains tested was observed. The presence of 20% human serum abolished the bactericidal activity of hBD-3 against gram-negative strains and reduced the activity of the peptide against gram-positive strains

The N-Terminus of Human Lactoferrin Displays Anti-biofilm Activity on Candida parapsilosis in Lumen Catheters

Candida parapsilosis is a major cause of hospital-acquired infection, often related to parenteral nutrition administered via catheters and hand colonization of health care workers, and its peculiar biofilm formation ability on plastic surfaces. The mortality rate of 30% points to the pressing need for new antifungal drugs. The present study aimed at analyzing the inhibitory activity of the N-terminal lactoferrin-derived peptide, further referred to as hLF 1-11, against biofilms produced by clinical isolates of C. parapsilosis characterized for their biofilm forming ability and fluconazole susceptibility. hLF 1-11 anti-biofilm activity was assessed in terms of reduction of biofilm biomass, metabolic activity, and observation of sessile cell morphology on polystyrene microtiter plates and using an in vitro model of catheter-associated C. parapsilosis biofilm production. Moreover, fluctuation in transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production upon peptide exposure were evaluated by quantitative real time RT-PCR. The results revealed that hLF 1-11 exhibits an inhibitory effect on biofilm formation by all the C. parapsilosis isolates tested, in a dose-dependent manner, regardless of their fluconazole susceptibility. In addition, hLF 1-11 induced a statistically significant dose-dependent reduction of preformed-biofilm cellular density and metabolic activity at high peptide concentrations only. Interestingly, when assessed in a catheter lumen, hLF 1-11 was able to induce a 2-log reduction of sessile cell viability at both the peptide concentrations used in RPMI diluted in NaPB. A more pronounced anti-biofilm effect was observed (3.5-log reduction) when a 10% glucose solution was used as experimental condition on both early and preformed C. parapsilosis biofilm. Quantitative real time RT-PCR experiments confirmed that hLF 1-11 down-regulates key biofilm related genes. The overall findings suggest hLF 1-11 as a promising candidate for the prevention of C. parapsilosis biofilm formation and to treatment of mature catheter-related C. parapsilosis biofilm formation

Involvement of the<i>Mycobacterium tuberculosis</i>secreted antigen SA-5K in intracellular survival of recombinant<i>Mycobacterium smegmatis</i>

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Impact of Mycobacterium tuberculosis RD1-locus on human primary dendritic cell immune functions

Modern strategies to develop vaccines against Mycobacterium tuberculosis (Mtb) aim to improve the current Bacillus Calmette-Guerin (BCG) vaccine or to attenuate the virulence of Mtb vaccine candidates. In the present study, the impact of wild type or mutated region of difference 1 (RD1) variants on the immunogenicity of Mtb and BCG recombinants was investigated in human primary dendritic cells (DC). A comparative analysis of transcriptome, signalling pathway activation, maturation, apoptosis, cytokine production and capacity to promote Th1 responses demonstrated that DC sense quantitative and qualitative differences in the expression of RD1-encoded factors - ESAT6 and CFP10 - within BCG or Mtb backgrounds. Expansion of IFN-γ producing T cells was promoted by BCG::RD1-challenged DC, as compared to their BCG-infected counterparts. Although Mtb recombinants acted as a strong Th-1 promoting stimulus, even with RD1 deletion, the attenuated Mtb strain carrying a C-terminus truncated ESAT-6 elicited a robust Th1 promoting phenotype in DC. Collectively, these studies indicate a necessary but not sufficient role for the RD1 locus in promoting DC immune-regulatory functions. Additional mycobacterial factors are likely required to endow DC with a high Th1 polarizing capacity, a desirable attribute for a successful control of Mtb infection

ESAT-6 Secretion-Independent Impact of ESX-1 Genes espF and espG1 on Virulence of Mycobacterium tuberculosis

Background. The pathogenesis of Mycobacterium tuberculosis largely depends on the secretion of the 6-kD early secreted antigenic target ESAT-6 (EsxA) and the 10-kD culture filtrate protein CFP-10 (EsxB) via the ESX-1/typeVII secretion system. Although gene products from the core RD1 region have been shown to be deeply implicated in this process, less is known about proteins encoded further upstream in the 5′ region of the ESX-1 cluster, such as the ESX-1 secretion-associated proteins (Esps) EspF or EspG1. Methods. To elucidate the role of EspF/G1, whose orthologs in Mycobacterium marinum and Mycobacterium smegmatis are reportedly involved in EsxA/B secretion, we constructed 3 M. tuberculosis knockout strains deleted for espF, espG1 or the segment corresponding to the combined RD1bcg-RD1mic region of bacille Calmette-Guérin (BCG) and Mycobacterium microti, which also contains espF and espG1. Results. Analysis of these strains revealed that, unlike observations with the model organisms M. smegmatis or M. marinum, disruption of espF and espG1 in M. tuberculosis did not impact the secretion and T cell recognition of EsxA/B but still caused severe attenuation. Conclusions. The separation of the 2 ESX-1-connected phenotypes (ie, EsxA/B secretion and virulence) indicates that EsxA/B secretion is not the only readout for a functional ESX-1 system and suggests that other processes involving EspF/G1 also play important roles in ESX-1-mediated pathogenicit

Control of M. tuberculosis ESAT-6 Secretion and Specific T Cell Recognition by PhoP

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra “knock-ins” that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis

Mycobacterium tuberculosis Exploits Asparagine to Assimilate Nitrogen and Resist Acid Stress during Infection

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes. © 2014 Gouzy et al

Systematic Genetic Nomenclature for Type VII Secretion Systems

CITATION: Bitter, W., et al. 2009. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathogens, 5(10): 1-6, doi: 10.1371/journal.ppat.1000507.The original publication is available at http://journals.plos.org/plospathogensMycobacteria, such as the etiological agent of human tuberculosis, Mycobacterium tuberculosis, are protected by an impermeable cell envelope composed of an inner cytoplasmic membrane, a peptidoglycan layer, an arabinogalactan layer, and an outer membrane. This second membrane consists of covalently linked, tightly packed long-chain mycolic acids [1,2] and noncovalently bound shorter lipids involved in pathogenicity [3–5]. To ensure protein transport across this complex cell envelope, mycobacteria use various secretion pathways, such as the SecA1-mediated general secretory pathway [6,7], an alternative SecA2-operated pathway [8], a twin-arginine translocation system [9,10], and a specialized secretion pathway variously named ESAT-6-, SNM-, ESX-, or type VII secretion [11–16]. The latter pathway, hereafter referred to as type VII secretion (T7S), has recently become a large and competitive research topic that is closely linked to studies of host–pathogen interactions of M. tuberculosis [17] and other pathogenic mycobacteria [16]. Molecular details are just beginning to be revealed [18–22] showing that T7S systems are complex machineries with multiple components and multiple substrates. Despite their biological importance, there has been a lack of a clear naming policy for the components and substrates of these systems. As there are multiple paralogous T7S systems within the Mycobacteria and orthologous systems in related bacteria, we are concerned that, without a unified nomenclature system, a multitude of redundant and obscure gene names will be used that will inevitably lead to confusion and hinder future progress. In this opinion piece we will therefore propose and introduce a systematic nomenclature with guidelines for name selection of new components that will greatly facilitate communication and understanding in this rapidly developing field of research.http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1000507Publisher's versio

Mycobacterial PE, PPE and ESX clusters: Novel insights into the secretion of these most unusual protein families

The human pathogen Mycobacterium tuberculosis harbours a large number of genes that encode proteins whose N-termini contain the characteristic motifs Pro-Glu (PE) or Pro-Pro-Glu (PPE). A subgroup of the PE proteins contains polymorphic GC-rich sequences (PGRS), while a subgroup of the PPE proteins contains major polymorphic tandem repeats (MPTR). The function of most of these proteins remains unknown. However, in this issue of Molecular Microbiology, Abdallah and colleagues show that PE-PGRS proteins from the model organism Mycobacterium marinum are secreted by components of the ESX-5 system that belongs to the recently defined type VII secretion systems. These observations, which now need to be addressed and confirmed in M. tuberculosis, open new perspectives on the function of these highly abundant proteins. © 2009 Blackwell Publishing Ltd