Intracellular Pathogenic Bacteria Research Articles

Essential functions linked with structural disorder in organisms of minimal genome.

Intrinsically disordered regions (IDRs) of proteins fulfill important regulatory roles in most organisms. However, the proteins of certain endosymbiont and intracellular pathogenic bacteria with extremely reduced genomes contain disproportionately small amounts of IDRs, consisting almost entirely of folded domains. As their genomes co-evolving with their hosts have been reduced in unrelated lineages, the proteomes of these bacteria represent independently evolved minimal protein sets. We systematically analyzed structural disorder in a representative set of such minimal organisms to see which types of functionally relevant longer IDRs are invariably retained in them. We found that a few characteristic functions are consistently linked with conformational disorder: ribosomal proteins, key components of the protein production machinery, a central coordinator of DNA metabolism and certain housekeeping chaperones seem to strictly rely on structural disorder even in genome-reduced organisms. We propose that these functions correspond to the most essential and probably also the most ancient ones fulfilled by structural disorder in cellular organisms.ReviewersThis article was reviewed by Michael Gromiha, Zoltan Gaspari and Sandor Pongor.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0149-y) contains supplementary material, which is available to authorized users.

Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

Phenotypic characterization of the Francisella tularensis ΔpdpC and ΔiglG mutants

Several bacterial pathogens interact with their host through protein secretion effectuated by a type VI secretion system (T6SS). Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. Proteins encoded by the Francisella pathogenicity island (FPI), which constitute a type VI secretion system, are essential for the virulence of the bacterium and a key mechanism behind this is the escape from the phagosome followed by productive cytosolic replication. It has been shown that T6SS in Francisella is distinct since all putative substrates of F. tularensis T6SS, except for VgrG, are unique to the species. Many of the FPI proteins are secreted into the macrophage cytosol and this is dependent on the functional components of DotU, VgrG, IglC and IglG. In addition, PdpC seems to have a regulatory role for the expression of iglABCD. Since previous results showed peculiar phenotypes of the ΔpdpC and ΔiglG mutants in mouse macrophages, their unique behavior was characterized in human monocyte-derived macrophages (HMDM) in this study. Our results show that both ΔpdpC and ΔiglG mutants of the live vaccine strain (LVS) of F. tularensis did not replicate within HMDMs. The ΔpdpC mutant did not escape from the Francisella containing phagosome (FCP), neither caused cytopathogenicity in primary macrophages and was attenuated in a mouse model. Interestingly, the ΔiglG mutant escaped from the HMDMs FCP and also caused pathological changes in the spleen and liver tissues of intradermally infected C57BL/6 mice. The ΔiglG mutant, with its unique phenotype, is a potential vaccine candidate.

Gold nanoparticle-DNA aptamer conjugate-assisted delivery of antimicrobial peptide effectively eliminates intracellular Salmonella enterica serovar Typhimurium.

Antimicrobial peptides (AMPs) are a promising new class of antibacterial compounds. However, their applications in the treatment of intracellular pathogenic bacteria have been limited by their invivo instability and low penetrating ability into mammalian cells. Here, we report that gold nanoparticles conjugated with DNA aptamer (AuNP-Apt) efficiently delivered AMPs into mammalian living systems with enhanced stability of the AMPs. C-terminally hexahistidine-tagged A3-APO (A3-APO(His)) AMPs were loaded onto AuNPs conjugated with His-tag DNA aptamer (AuNP-Apt(His)) by simple mixing and were delivered into Salmonella enterica serovar Typhimurium (S. Typhimurium)-infected HeLa cells, resulting in the increased viability of host cells due to the elimination of intracellular S. Typhimurium cells. Furthermore, the intravenous injection of AuNP-Apt(His) loaded with A3-APO(His) into S. Typhimurium-infected mice resulted in a complete inhibition of S. Typhimurium colonization in the mice organs, leading to 100% survival of the mice. Therefore, AuNP-Apt(His) can serve as an innovative platform for AMP therapeutics to treat intracellular bacterial infections in mammals.

Computer-aided vaccine designing approach against fish pathogens Edwardsiella tarda and Flavobacterium columnare using bioinformatics softwares.

Edwardsiella tarda and Flavobacterium columnare are two important intracellular pathogenic bacteria that cause the infectious diseases edwardsiellosis and columnaris in wild and cultured fish. Prediction of major histocompatibility complex (MHC) binding is an important issue in T-cell epitope prediction. In a healthy immune system, the T-cells must recognize epitopes and induce the immune response. In this study, T-cell epitopes were predicted by using in silico immunoinformatics approach with the help of bioinformatics tools that are less expensive and are not time consuming. Such identification of binding interaction between peptides and MHC alleles aids in the discovery of new peptide vaccines. We have reported the potential peptides chosen from the outer membrane proteins (OMPs) of E. tarda and F. columnare, which interact well with MHC class I alleles. OMPs from E. tarda and F. columnare were selected and analyzed based on their antigenic and immunogenic properties. The OMPs of the genes TolC and FCOL_04620, respectively, from E. tarda and F. columnare were taken for study. Finally, two epitopes from the OMP of E. tarda exhibited excellent protein–peptide interaction when docked with MHC class I alleles. Five epitopes from the OMP of F. columnare had good protein–peptide interaction when docked with MHC class I alleles. Further in vitro studies can aid in the development of potential peptide vaccines using the predicted peptides.

Maternal vaccination with dPNAG protects foals against the intracellular pathogen Rhodococcus equi

Abstract Rhodococcus equi is a facultative intracellular pathogenic bacterium that causes a form of pneumonia similar to tuberculosis in foals and immunocompromised humans. A vaccine against R. equi is lacking. We studied the efficacy of maternal vaccination with deacetylated poly-N-acetyl glucosamine (dPNAG) oligosaccharide conjugated to tetanus toxoid to protect foals against R. equi. Pregnant mares (n=9) were randomly assigned to intramuscular injection 6 and 3 weeks prior to foaling with either vaccine (n=5) or saline (n=4). Foals were infected intrabronchially at age 28 days with 1 × 106 of a single strain of live, virulent R. equi. Foals were examined twice daily for clinical signs of R. equi pneumonia (viz., coughing, tachypnea, dyspnea, temperature >103°F), and weekly by thoracic ultrasonography. Blood samples were collected at age 2, 14, 28, 42, 56, and 84 days from all foals to detect antibodies by ELISA against dPNAG and native PNAG in serum and IFNγ production from peripheral blood mononuclear cells (PBMCs). The proportion of foals with pneumonia was significantly (P<0.05) greater among the unvaccinated mares (3/4) than vaccinated mares (0/5). Titers of dPNAG and PNAG antibodies were significantly (P<0.05) higher at all ages for foals of vaccinated mares. IFNγ produced by PBMCs stimulated with R. equi antigen was significantly greater (P<0.05) at age 28 days (prior to infection) in foals of vaccinated mares. Results indicate the dPNAG vaccine protected foals against the intracellular pathogen R. equi and efficacy against R. equi was likely mediated by maternally-transferred antibodies and enhanced CMI responses, and contribute to a growing body of evidence for a key role of antibodies in protecting against intracellular pathogens.

Loss of Conserved Noncoding RNAs in Genomes of Bacterial Endosymbionts.

The genomes of intracellular symbiotic or pathogenic bacteria, such as of Buchnera, Mycoplasma, and Rickettsia, are typically smaller compared with their free-living counterparts. Here we showed that noncoding RNA (ncRNA) families, which are conserved in free-living bacteria, frequently could not be detected by computational methods in the small genomes. Statistical tests demonstrated that their absence is not an artifact of low GC content or small deletions in these small genomes, and thus it was indicative of an independent loss of ncRNAs in different endosymbiotic lineages. By analyzing the synteny (conservation of gene order) between the reduced and nonreduced genomes, we revealed instances of protein-coding genes that were preserved in the reduced genomes but lost cis-regulatory elements. We found that the loss of cis-regulatory ncRNA sequences, which regulate the expression of cognate protein-coding genes, is characterized by the reduction of secondary structure formation propensity, GC content, and length of the corresponding genomic regions.

Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria.

The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701) after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages.

The Mutualistic Side of Wolbachia-Isopod Interactions: Wolbachia Mediated Protection Against Pathogenic Intracellular Bacteria.

Wolbachia is a vertically transmitted endosymbiont whose radiative success is mainly related to various host reproductive manipulations that led to consider this symbiont as a conflictual reproductive parasite. However, lately, some Wolbachia have been shown to act as beneficial symbionts by protecting hosts against a broad range of parasites. Still, this protection has been mostly demonstrated in artificial Wolbachia-host associations between partners that did not co-evolved together. Here, we tested in two terrestrial isopod species Armadillidium vulgare and Porcellio dilatatus whether resident Wolbachia (native or non-native) could confer protection during infections with Listeria ivanovii and Salmonella typhimurium and also during a transinfection with a Wolbachia strain that kills the recipient host (i.e., wVulC in P. dilatatus). Survival analyses showed that (i) A. vulgare lines hosting their native Wolbachia (wVulC) always exhibited higher survival than asymbiotic ones when infected with pathogenic bacteria (ii) P. dilatatus lines hosting their native wDil Wolbachia strain survived the S. typhimurium infection better, while lines hosting non-native wCon Wolbachia strain survived the L. ivanovii and also the transinfection with wVulC from A. vulgare better. By studying L. ivanovii and S. typhimurium loads in the hemolymph of the different host-Wolbachia systems, we showed that (i) the difference in survival between lines after L. ivanovii infections were not linked to the difference between their pathogenic bacterial loads, and (ii) the difference in survival after S. typhimurium infections corresponds to lower loads of pathogenic bacteria. Overall, our results demonstrate a beneficial effect of Wolbachia on survival of terrestrial isopods when infected with pathogenic intracellular bacteria. This protective effect may rely on different mechanisms depending on the resident symbiont and the invasive bacteria interacting together within the hosts.

Bacterial small RNAs in the Genus Rickettsia.

BackgroundRickettsia species are obligate intracellular Gram-negative pathogenic bacteria and the etiologic agents of diseases such as Rocky Mountain spotted fever (RMSF), Mediterranean spotted fever, epidemic typhus, and murine typhus. Genome sequencing revealed that R. prowazekii has ~25 % non-coding DNA, the majority of which is thought to be either “junk DNA” or pseudogenes resulting from genomic reduction. These characteristics also define other Rickettsia genomes. Bacterial small RNAs, whose biogenesis is predominantly attributed to either the intergenic regions (trans-acting) or to the antisense strand of an open reading frame (cis-acting), are now appreciated to be among the most important post-transcriptional regulators of bacterial virulence and growth. We hypothesize that intergenic regions in rickettsial species encode for small, non-coding RNAs (sRNAs) involved in the regulation of its transcriptome, leading to altered virulence and adaptation depending on the host niche.ResultsWe employed a combination of bioinformatics and in vitro approaches to explore the presence of sRNAs in a number of species within Genus Rickettsia. Using the sRNA Identification Protocol using High-throughput Technology (SIPHT) web interface, we predicted over 1,700 small RNAs present in the intergenic regions of 16 different strains representing 13 rickettsial species. We further characterized novel sRNAs from typhus (R. prowazekii and R. typhi) and spotted fever (R. rickettsii and R. conorii) groups for their promoters and Rho-independent terminators using Bacterial Promoter Prediction Program (BPROM) and TransTermHP prediction algorithms, respectively. Strong σ70 promoters were predicted upstream of all novel small RNAs, indicating the potential for transcriptional activity. Next, we infected human microvascular endothelial cells (HMECs) with R. prowazekii for 3 h and 24 h and performed Next Generation Sequencing to experimentally validate the expression of 26 sRNA candidates predicted in R. prowazekii. Reverse transcriptase PCR was also used to further verify the expression of six putative novel sRNA candidates in R. prowazekii.ConclusionsOur results yield clear evidence for the expression of novel R. prowazekii sRNA candidates during infection of HMECs. This is the first description of novel small RNAs for a highly pathogenic species of Rickettsia, which should lead to new insights into rickettsial virulence and adaptation mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2293-7) contains supplementary material, which is available to authorized users.

Toward an optimized treatment of intracellular bacterial infections: input of nanoparticulate drug delivery systems.

Intracellular pathogenic bacteria can lead to some of the most life-threatening infections. By evolving a number of ingenious mechanisms, these bacteria have the ability to invade, colonize and survive in the host cells in active or latent forms over prolonged period of time. A variety of nanoparticulate systems have been developed to optimize the delivery of antibiotics. Main advantages of nanoparticulate systems as compared with free drugs are an efficient drug encapsulation, protection from inactivation, targeting infection sites and the possibility to deliver drugs by overcoming cellular barriers. Nevertheless, despite the great progresses in treating intracellular infections using nanoparticulate carriers, some challenges still remain, such as targeting cellular subcompartments with bacteria and delivering synergistic drug combinations. Engineered nanoparticles should allow controlling drug release both inside cells and within the extracellular space before reaching the target cells.

ID: 224: Stat2-dependent responses lead to increased mortality following Salmonella-induced colitis

Salmonella Typhimurium is an intracellular pathogenic gram-negative bacterium that colonizes predominantly the intestinal lumen, activates Toll-like receptors, and accounts for most of food borne illnesses. The transcription factor Stat2 is vital in type I interferon activated antiviral and antitumor immunity and yet less is known about its role in bacterial infection. In a recent study, we reported that Stat2 was protective when tested in a mouse model of lipopolysaccharide-induced sepsis as mortality was accelerated inStat2 knockout (KO) mice. In this study, we evaluated the effect of Stat2 in a clinically relevant inflammation model of Salmonella-induced colitis. We found that when mice were pretreated with streptomycin and then orally infected with Salmonella, compared to wild type mice, Stat2KO mice showed altered intestinal responses by experiencing lower intestinal bacterial burdens. No differences were found in bacterial burdens at systemic sites. Stat2-dependent responses were lethal to wild type mice while Stat2KO mice were found to be more resistant. In contrast, Stat1KO mice were more susceptible to Salmonella infection than wild type mice suggesting that Stat2 and Stat1 play different roles during pathogenic bacterial infection. Our results indicate that disruption of the intestinal microbiota allows Stat2-dependent responses to play an adverse role in host defense against Salmonella-induced colitis. To our knowledge, this is the first evidence of Stat2 dependent responses playing a negative role in the host defense against pathogenic Salmonella bacteria. Future studies will reveal if this observation applies to other gram negative pathogens.

Legionella Pathogenesis and Virulence Factors

Legionella is the causative agent of legionellosis or Legionnaires’disease. Infection with Legionella spp. is an important cause of community and hospital-acquired pneumonia. Infection with Legionella is also one of the most common causes of severe pneumonia in community settings, and Legionella is isolated in 40% of hospital-acquired pneumonia cases. Intense research on Legionella pneumophila (L. pneumophila) and other Legionella spp. over the last two decades provided insights into the evolution, ecology, epidemiology, and pathogenesis of this opportunistic pathogenic and facultative intracellular bacterium. An in-depth understanding of the ecology and virulence factors of Legionella spp. will contribute to an in-depth comprehension of its pathogenesis and efficient, sustainable elimination of the bacteria from anthropogenic water systems and might lead to the development of novel therapeutics. This review summarizes available information regarding the cellular microbiology, pathogenesis and virulence factors of Legionella spp. with an emphasis on the potential virulence factors and the contribution of these factors to the pathogenesis.

TLR2 Signaling is Required for the Innate, but Not Adaptive Response to LVS clpB.

Toll-like receptor 2 (TLR2) is the best-characterized pattern-recognition receptor for the highly pathogenic intracellular bacterium, Francisella tularensis. We previously identified a mutant in the live vaccine strain (LVS) of Francisella, LVS clpB, which is attenuated, but induces a protective immune response. We sought to determine whether TLR2 signaling was required during the immune response to LVS clpB. TLR2 knock-out (TLR2 KO) mice previously infected with LVS clpB are completely protected during a lethal challenge with LVS. Furthermore, the kinetics and magnitude of the primary T-cell response in B6 and TLR2 KO mice are similar indicating that TLR2 signaling is dispensable for the adaptive immune response to LVS clpB. TLR2 signaling was important, however, for the innate immune response to LVS clpB. We identified three classes of cytokines/chemokines that differ in their dependence on TLR2 signaling for production on day 3 post-inoculation in the bronchoalveolar lavage fluid. IL-1α, IL-1β, IL-2, IL-17, MIP-1α, and TNF-α production depended on TLR2 signaling, while GM-CSF, IFN-γ, and VEGF production were completely independent of TLR2 signaling. IL-6, IL-12, IP-10, KC, and MIG production were partially dependent on TLR2 signaling. Together our data indicate that the innate immune response to LVS clpB requires TLR2 signaling for the maximal innate response, whereas TLR2 is not required for the adaptive immune response.

Effector bottleneck: microbial reprogramming of parasitized host cell transcription by epigenetic remodeling of chromatin structure.

Obligate intracellular pathogenic bacteria evolved to manipulate their host cells with a limited range of proteins constrained by their compact genomes. The harsh environment of a phagocytic defense cell is one that challenges the majority of commensal and pathogenic bacteria; yet, these are the obligatory vertebrate homes for important pathogenic species in the Anaplasmataceae family. Survival requires that the parasite fundamentally alter the native functions of the cell to allow its entry, intracellular replication, and transmission to a hematophagous arthropod. The small genomic repertoires encode several eukaryotic-like proteins, including ankyrin A (AnkA) of Anaplasma phagocytophilum and Ank200 and tandem-repeat containing proteins of Ehrlichia chaffeensis that localize to the host cell nucleus and directly bind DNA. As a model, A. phagocytophilum AnkA appears to directly alter host cell gene expression by recruiting chromatin modifying enzymes such as histone deacetylases and methyltransferases or by acting directly on transcription in cis. While cis binding could feasibly alter limited ranges of genes and cellular functions, the complex and dramatic alterations in transcription observed with infection are difficult to explain on the basis of individually targeted genes. We hypothesize that nucleomodulins can act broadly, even genome-wide, to affect entire chromosomal neighborhoods and topologically associating chromatin domains by recruiting chromatin remodeling complexes or by altering the folding patterns of chromatin that bring distant regulatory regions together to coordinate control of transcriptional reprogramming. This review focuses on the A. phagocytophilum nucleomodulin AnkA, how it impacts host cell transcriptional responses, and current investigations that seek to determine how these multifunctional eukaryotic-like proteins facilitate epigenetic alterations and cellular reprogramming at the chromosomal level.

How bacterial effectors hijack small GTPases to divert membrane traffic

Membrane traffic, which is the "cellular postal service" that shuttles biomolecules around the cell and organizes the structure of organelles, is among the primary targets of effectors injected by intracellular pathogenic bacteria to invade their host and avoid from being destroyed. I will present our recent structural and biochemical studies of effectors from Legionella pneumophila (the bacteria that causes the legionnaire's disease, a severe pneumonia) that divert membrane traffic to generate a membrane-bound vacuole where the pathogen hides and replicates. One of these effectors, AnkX, is a FIC domain-containing toxin that alters the functions of a Rab GTPase involved in vesicular traffic at the endoplasmic reticulum, by covalent attachment of a phosphocholine molecule. The other one, RalF, functions as an illegitimate guanine nucleotide exchange factor to activate an Arf GTPase on the vacuole. Our studies showed how AnkX binds and processes CDP-choline to transfer phosphocholine onto Rab1 [1], and uncover a novel membrane sensor in RalF that controls its localization and activity [2]

Polymorphism in gyrA is associated to quinolones resistance in Chilean Piscirickettsia salmonis field isolates.

Keywords: antibiotic resistance, gyrA, Piscirickettsiasalmonis, quinolones.Piscirickettsia salmonis is a gram-negative, pleomor-phic coccoid-shaped and facultative intracellularfish pathogenic bacterium, that causes piscirickettsi-osis, a disease also known as salmonid rickettsialsepticaemia (SRS) (Fryer et al. 1990). In Chile,P. salmonis was isolated for the first time in 1989,but probably has been present since 1983 (Bravo C Rozas &Enriquez 2013). SRS has also been reported inimportant salmon producing countries such asCanada, Ireland, Scotland and Norway, but with alower economic impact (Rozas & Enriquez 2013).P. salmonis cells are slow growing and hard to culti-vate in free-cell media, which has hindered its isola-tion and further characterization. However, recentlydescribed supplemented media (Mikalsen et al.2008) facilitate their growth and the antibiotictesting assays to evaluate effective antimicrobial ther-apy. In this regard, quinolones were the former mostwidely used antimicrobial compounds in Chileanaquaculture, especially as a first line drug againstSRS.In Chile, a large proportion of antibiotics is usedfor prophylaxis rather than for chemotherapy. Forinstance, in comparison with Norway and Canada,1500 and eight times more antimicrobials wereneeded to obtain similar amounts of salmon inChile in 2007 (Cabello et al. 2013). The same year,930 metric tons of antibiotics were mostly used insalmon production. Of those antibiotics, 24% werequinolones (Cabello et al. 2013). This practice hasalready altered the microbiota of sediments in closeproximity to aquaculture sites, where a significantfraction of isolated bacteria is antibiotic resistant(Buschmann et al. 2012). Therefore, the emer-gence and potential spread of antibiotic-resistantP. salmonis isolates cannot be ruled out.Twenty P. salmonis isolates cultured from kid-ney samples of diseased fish collected from diversegeographic origin (Fig. 1) between 2010 and2012 were phenotypically characterized for quinol-ones susceptibility. The isolates were grown onagar (Mikalsen et al. 2008), at 18 °C for 10 daysand kept frozen in liquid nitrogen. For antibioticsensitivity testing, the minimal inhibitory concen-tration (MIC) for enrofloxacin, flumequine andoxolinic acid were assessed according to theinstructions given by the Clinical and LaboratoryStandards Institute (CLSI), guide M49-A. 96-wellmicroplates containing twofold serial dilutions(0.03–64 lgmL

Structure and mechanism of bacterial effectors that hijack trafficking small GTPases (LB278)

Membrane traffic, which is the “cellular postal service” that shuttles biomolecules around the cell and organizes the structure of organelles, is among the primary targets of effectors injected by intracellular pathogenic bacteria to invade their host and avoid from being destroyed. We will present our recent structural and biochemical studies of effectors from Legionella pneumophila (the bacteria that causes the legionnaire’s disease, a severe pneumonia) which divert membrane traffic to generate a membrane‐bound vacuole where the pathogen hides and replicates. One of these effectors, AnkX, is a FIC domain‐containing toxin that alters the functions of a Rab GTPase involved in vesicular traffic at the endoplasmic reticulum, by covalent attachment of a phosphocholine molecule. The other one, RalF, functions as an illegitimate guanine nucleotide exchange factor to illegitimately activate an Arf GTPase on the vacuole. Our studies showed how AnkX binds and process CDP‐choline to transfer phosphocholine to Rab1 [1], and uncover a novel membrane sensor in RalF that controls its localization and activity [2] [1] Campanacci V, Mukherjee S, Roy CR, Cherfils J. The mechanism of phosphocholine transfer by AnkX, an ankyrin repeats and FIC domain‐containing Legionella effector. EMBO J. (2013) 32:1469‐77.[2] A Novel Membrane Sensor Controls the Localization and ArfGEF Activity of Bacterial RalF. Folly‐Klan M, Alix E, Stalder D, Ray P, Duarte LV, Delprato A, Zeghouf M, Antonny B, Campanacci V, Roy CR, Cherfils J. PLoS Pathog. (2013) 9(11):e1003747.Grant Funding Source: Supported by CNRS and ANR

Shigella Type III Secretion Protein MxiI Is Recognized by Naip2 to Induce Nlrc4 Inflammasome Activation Independently of Pkcδ

Recognition of intracellular pathogenic bacteria by members of the nucleotide-binding domain and leucine-rich repeat containing (NLR) family triggers immune responses against bacterial infection. A major response induced by several Gram-negative bacteria is the activation of caspase-1 via the Nlrc4 inflammasome. Upon activation, caspase-1 regulates the processing of proIL-1β and proIL-18 leading to the release of mature IL-1β and IL-18, and induction of pyroptosis. The activation of the Nlrc4 inflammasome requires the presence of an intact type III or IV secretion system that mediates the translocation of small amounts of flagellin or PrgJ-like rod proteins into the host cytosol to induce Nlrc4 activation. Using the Salmonella system, it was shown that Naip2 and Naip5 link flagellin and the rod protein PrgJ, respectively, to Nlrc4. Furthermore, phosphorylation of Nlrc4 at Ser533 by Pkcδ was found to be critical for the activation of the Nlrc4 inflammasome. Here, we show that Naip2 recognizes the Shigella T3SS inner rod protein MxiI and induces Nlrc4 inflammasome activation. The expression of MxiI in primary macrophages was sufficient to induce pyroptosis and IL-1β release, which were prevented in macrophages deficient in Nlrc4. In the presence of MxiI or Shigella infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited Shigella-induced caspase-1 activation, IL-1β maturation and Asc pyroptosome formation. Notably, the Pkcδ kinase was dispensable for caspase-1 activation and secretion of IL-1β induced by Shigella or Salmonella infection. These results indicate that activation of caspase-1 by Shigella is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkcδ kinase.

Effect of host factors on neutrophil functions in response to Burkholderia pseudomallei in healthy Thai subjects.

Burkholderia pseudomallei is an intracellular pathogenic bacterium that causes melioidosis in humans. On infection, neutrophils eliminate the majority of intracellular B. pseudomallei. Previous reports on the risk factors for melioidosis have shown that host factors, particularly age and diabetes mellitus, increase susceptibility to B. pseudomallei; however, whether these factors influence neutrophil functions in response to infection remains unknown. In this study, whole blood samples were collected from healthy Thai blood donors and co-cultured with B. pseudomallei, and phagocytic and respiratory burst functions of neutrophils were then measured by flow cytometry. The results show reduced neutrophil functions in older donors or those with poor glycemic control. Furthermore, the levels of antibody against B. pseudomallei showed a positive correlation with neutrophil functions. This study therefore indicated the importance of age, glycemic control, and antibody levels in the activity of neutrophils in melioidosis.