Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

Effects of submaximal and supramaximal interval training on determinants of endurance performance in endurance athletes.

We compared the effects of submaximal and supramaximal cycling interval training on determinants of exercise performance in moderately endurance-trained men. Maximal oxygen consumption (VO2max ), peak power output (Ppeak ), and peak and mean anaerobic power were measured before and after 6 weeks (3 sessions/week) of submaximal (85% maximal aerobic power [MP], HIIT85 , n = 8) or supramaximal (115% MP, HIIT115 , n = 9) interval training to exhaustion in moderately endurance-trained men. High-intensity training volume was 47% lower in HIIT115 vs HIIT85 (304 ± 77 vs 571 ± 200 min; P < 0.01). Exercise training was generally associated with increased VO2max (HIIT85 : +3.3 ± 3.1 mL/kg/min; HIIT115 : +3.3 ± 3.6 ml/kg/min; Time effect P = 0.002; Group effect: P = 0.95), Ppeak (HIIT85 : +18 ± 9 W; HIIT115 : +16 ± 27 W; Time effect P = 0.045; Group effect: P = 0.49), and mean anaerobic power (HIIT85 : +0.42 ± 0.69 W/kg; HIIT115 : +0.55 ± 0.65 W/kg; Time effect P = 0.01; Group effect: P = 0.18). Six weeks of submaximal and supramaximal interval training performed to exhaustion seems to equally improve VO2max and anaerobic power in endurance-trained men, despite half the accumulated time spent at the target intensity.

Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut.

The mammalian gastrointestinal tract is colonized by a high-density polymicrobial community where bacteria compete for niches and resources. One key competition strategy includes cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), a multiprotein needle-like apparatus that injects effector proteins into prokaryotic and/or eukaryotic target cells. However, the contribution of T6SS antibacterial activity during pathogen invasion of the gut has not been demonstrated. We report that successful establishment in the gut by the enteropathogenic bacterium Salmonella enterica serovar Typhimurium requires a T6SS encoded within Salmonella pathogenicity island-6 (SPI-6). In an in vitro setting, we demonstrate that bile salts increase SPI-6 antibacterial activity and that S Typhimurium kills commensal bacteria in a T6SS-dependent manner. Furthermore, we provide evidence that one of the two T6SS nanotube subunits, Hcp1, is required for killing Klebsiella oxytoca in vitro and that this activity is mediated by the specific interaction of Hcp1 with the antibacterial amidase Tae4. Finally, we show that K. oxytoca is killed in the host gut in an Hcp1-dependent manner and that the T6SS antibacterial activity is essential for Salmonella to establish infection within the host gut. Our findings provide an example of pathogen T6SS-dependent killing of commensal bacteria as a mechanism to successfully colonize the host gut.

The T6SSs of Pseudomonas aeruginosa Strain PAO1 and Their Effectors: Beyond Bacterial-Cell Targeting.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases such as chronic lung colonization in cystic fibrosis patients and acute infections in hospitals. The capacity of P. aeruginosa to be pathogenic toward several hosts is notably due to different secretion systems. Amongst them, P. aeruginosa encodes three Type Six Secretion Systems (T6SS), named H1- to H3-T6SS, that act against either prokaryotes and/or eukaryotic cells. They are independent from each other and inject diverse toxins that interact with different components in the host cell. Here we summarize the roles of these T6SSs in the PAO1 strain, as well as the toxins injected and their targets. While H1-T6SS is only involved in antiprokaryotic activity through at least seven different toxins, H2-T6SS and H3-T6SS are also able to target prokaryotic as well as eukaryotic cells. Moreover, recent studies proposed that H2- and H3-T6SS have a role in epithelial cells invasion by injecting at least three different toxins. The diversity of T6SS effectors is astounding and other effectors still remain to be discovered. In this review, we present a table with other putative P. aeruginosa strain PAO1 T6SS-dependent effectors. Altogether, the T6SSs of P. aeruginosa are important systems that help fight other bacteria for their ecological niche, and are important in the pathogenicity process.

Genome-wide Screen of Pseudomonas aeruginosa in Saccharomyces cerevisiae Identifies New Virulence Factors.

Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. Fifty-one candidates were selected in athree-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec). By testing the cytotoxicity of wild type P. aeruginosa vs. pec mutants toward macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.

UNLABELLED: Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

Gene transfer: conjugation.

Conjugation is a gene transfer process in which a recipient bacterium receives DNA from a donor bacterium by cell-to-cell contact through conjugative pili. Conjugation is mediated by certain plasmids or transposons. Here, we describe plasmid conjugation.

The target cell genus does not matter.

Two type VI secreted phospholipases D of Pseudomonas aeruginosa were identified as trans-kingdom virulence effectors, targeting both prokaryotic and eukaryotic host cells. Each of them triggers killing bacterial competitors and internalization into non-phagocytic cells. These type VI lipolytic enzymes are widely distributed among pathogens and may constitute a conserved strategy.

[N-methyl-D-aspartate receptor antibody encephalitis: value of immunomodulatory therapy]. / Encéphalite à anticorps anti-récepteurs N-méthyl-D-aspartate (NMDA-R) : place des immunomodulateurs.

INTRODUCTION: Anti-N-methyl-D-aspartate receptor (NMDA-R) encephalitis is little known to pediatricians and likely underdiagnosed. The child's vital and cognitive prognosis is at stake. The use of immunomodulatory drugs, such as rituximab has led to spectacular results, but many questions remain about its mode of action in this type of pathology. CASE REPORT: We report the case of a 6-year-old girl with no medical history, admitted for status epilepticus preceded by behavior symptoms and sleep disorders. Gradually, the child became bedridden, mute, and animated by predominantly orofacial dyskinesia. Examinations were normal (cerebrospinal fluid [CSF] analysis, brain MRI). The diagnosis was established by the presence of NMDA-R antibodies in the CSF. After exclusion of a tumor-associated syndrome, treatment was started initially by intravenous immunoglobulins, then by plasma exchange, and finally rituximab. The patient was cured with rituximab despite an unusually early recovery of the B-cell pool. DISCUSSION: Anti-N-methyl-D-aspartate receptor (NMDA-R) encephalitis is a severe but potentially reversible neurologic disorder only recently described, even in childhood. It may be reversible without sequelae if diagnosed and treated early. The use of immunomodulatory therapy, such as rituximab seemingly improves the outcome. Immunological monitoring is needed to better understand its mechanism of action in autoimmune diseases of the nervous system in childhood.

Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa.

Three Type VI Secretion System (T6SS) loci called H1- to H3-T6SS coexist in Pseudomonas aeruginosa. H1-T6SS targets prokaryotic cells whereas H2-T6SS mediates interactions with both eukaryotic and prokaryotic host cells. Little is known about the third system, except that it may be connected to H2-T6SS during the host infection. Here we show that H3-T6SS is required for P. aeruginosa PAO1 virulence in the worm model. We demonstrate that the two putative H3-T6SS operons, called "left" and "right", are coregulated with H2-T6SS by the Las and Rhl Quorum Sensing systems. Interestingly, the RpoN σ54 factor has divergent effects on the three operons. As for many T6SSs, RpoN activates the expression of H3-T6SS left. However, RpoN unexpectedly represses the expression of H3-T6SS right and also H2-T6SS. Sfa2 and Sfa3 are putative enhancer binding proteins encoded on H2-T6SS and H3-T6SS left. In other T6SSs EBPs can act as σ54 activators to promote T6SS transcription. Strikingly, we found that the RpoN effects of H3-T6SS are Sfa-independent while the RpoN mediated repression of H2-T6SS is Sfa2-dependent. This is the first example of RpoN repression of a T6SS being mediated by a T6SS-encoded EBP.

Rituximab-induced T cell depletion in patients with rheumatoid arthritis: association with clinical response.

OBJECTIVE: Rituximab, a monoclonal antibody specifically targeting CD20, induces B cell depletion and is effective in the treatment of rheumatoid arthritis (RA). This study was undertaken to evaluate whether routine monitoring of lymphocyte subpopulations, especially T cells, may be useful in patients receiving rituximab for RA. METHODS: We examined data on all RA patients receiving rituximab between July 2007 and November 2012 in our center. Peripheral blood CD3+, CD4+, CD8+, CD3-CD56+, and CD19+ lymphocyte counts before and during the first course of rituximab were measured by flow cytometry. The Mann-Whitney nonparametric test was used to compare lymphocyte subpopulation counts before and during treatment. RESULTS: Data on 52 patients were examined. Rituximab induced unexpected and substantial depletion of T cells, mainly CD4+ cells, in most patients. The CD4+ cell count decreased by a mean ± SD of 37 ± 33% as compared to baseline at week 12, reaching <200 cells/µl in 3 patients. Importantly, lack of CD4+ cell depletion was associated with no clinical response. Therefore, the mechanism of action of rituximab may depend at least in part on T cells. CONCLUSION: Rituximab induces substantial T cell depletion, mainly of CD4+ cells, which is associated with the clinical response in RA. Routine monitoring of T cells may be useful in the clinical setting of RA.

CMV infection in the donor and increased kidney graft loss: impact of full HLA-I mismatch and posttransplantation CD8(+) cell reduction.

Despite a large body of literature, the impact of chronic cytomegalovirus (CMV) infection in donor on long-term graft survival remains unclear, and factors modulating the effect of CMV infection on graft survival are presently unknown. In this retrospective study of 1279 kidney transplant patients, we analyzed long-term graft survival and evolution of CD8(+) cell population in donors and recipients by CMV serology and antigenemia status. A positive CMV serology in the donor was an independent risk factor for graft loss, especially among CMV-positive recipients (R(+) ). Antigenemia was not a risk factor for graft loss and kidneys from CMV-positive donors remained associated with poor graft survival among antigenemia-free recipients. Detrimental impact of donor's CMV seropositivity on graft survival was restricted to patients with full HLA-I mismatch, suggesting a role of CD8(+) cells. In R(+) patients with positive CMV antigenemia during the first year, CD8(+) cell count did not increase at 2 years posttransplantation, in contrast to R(-) recipients. In addition, marked CD8(+) -cell decrease was a risk factor of graft failure in these patients. This study identifies HLA-I full mismatch and a decrease of CD8(+) cell count at 2 years as important determinants of CMV-associated graft loss.

The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells.

The genome of Pseudomonas aeruginosa PAO1 contains three type VI secretion systems (T6SSs) called H1-, H2-, and H3-T6SS. The H1-T6SS secretes three identified toxins that target other bacteria, providing a fitness advantage for P. aeruginosa, and likely contributes to bacterial pathogenesis in chronic infections. However, no specific substrates or defined roles have been described for the two other systems. Here, we demonstrate that the expression of H2-T6SS genes of strain PAO1 is up-regulated during the transition from exponential to stationary phase growth and regulated by the Las and Rhl quorum sensing systems. In addition, we identify two putative Fur boxes in the promoter region and find that H2-T6SS transcription is negatively regulated by iron. We also show that the H2-T6SS system enhances bacterial uptake into HeLa cells (75% decrease in internalization with a H2-T6SS mutant) and into lung epithelial cells through a phosphatidylinositol 3-kinase-dependent pathway that induces Akt activation in the host cell (50% decrease in Akt phosphorylation). Finally, we show that H2-T6SS plays a role in P. aeruginosa virulence in the worm model. Thus, in contrast to H1-T6SS, H2-T6SS modulates interaction with eukaryotic host cells. Together, T6SS can carry out different functions that may be important in establishing chronic P. aeruginosa infections in the human host.

Pre-treatment lymphopenia as a prognostic biomarker in colorectal cancer patients receiving chemotherapy.

BACKGROUND: Lymphopenia is a predictor of the efficacy and hematological toxicity of chemotherapy in various advanced cancers. There is little data about this relationship in colorectal cancer. In this retrospective study, the influence of pretreatment lymphopenia on hematological toxicity and the efficacy of chemotherapy was investigated in colorectal cancer patients. PATIENTS AND METHODS: In total, 260 patients were included in the study. Correlations between pre-treatment lymphopenia (lymphocyte count < 1,000/µl) and the occurrence of hematological toxicity and efficacy of first-line palliative chemotherapy were investigated. RESULTS: Lymphopenia was found in 49/260 (19%) patients. Ten of these patients with lymphopenia (20.4%) experienced severe hematological toxicity compared with 17 of the remaining 211 (8%) patients (P = 0.01). Lymphopenia was identified as an independent factor for hematological toxicity. Among patients who received palliative chemotherapy, the objective response rate was significantly lower in lymphopenic patients than in the other patients (12.5% vs. 40.2%; P = 0.004). Lymphopenia was strongly associated with shorter progression-free survival (median 4 vs. 7 months; P = 0.033) and shorter overall survival (median 16 vs. 24 months, P = 0.024). Multivariate analysis revealed that lymphopenia had an independent effect on survival. CONCLUSIONS: Our findings show that lymphopenia is an independent predictive factor for both hematological toxicity and efficacy of chemotherapy in colorectal cancer. Pre-treatment lymphocyte count may represent a simple and new predictive biomarker of chemotherapy effects in colorectal cancer patients.

[Implications of receptors for the Fc portion of IgG (FcgammaRs) in mechanism of action of therapeutic antibodies]. / Rôle des récepteurs à la portion Fc des IgG (FcgammaRs) dans l'activité des anticorps thérapeutiques.

From several years ago, recombinant monoclonal antibodies have allowed a revolution in therapeutic approach of cancer patients. Whereas the clinical efficacy of many antibodies is now demonstrated, their mechanism of action in patients remains elusive. For antibodies targeting membrane antigens, they particularly resort to cytotoxic effectors, which expressed receptors for Fc portion of IgG (FcgammaRs). This review analyses different functions depending of FcgammaR and their potential role in mechanism of action of therapeutic antibodies. A better knowledge of these functions should allow in the next future the optimisation of these treatments.