The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon—Shigella IpaC, Salmonella SipC, or Chromobacterium CipC—determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.Intracellular bacterial pathogens use a variety of elaborate means to survive within host cells. Postinvasion, some such as Legionella, Salmonella, and Chlamydia species modify bacteria-containing vacuoles to avoid death via phagosomal acidification or lysosomal fusion. Others, including Shigella, Listeria, Rickettsia, and Burkholderia species, rapidly escape from phagosomes into the cytosol of infected cells. Although escape from phagosomes by the classic intracytoplasmic Gram-positive bacterium Listeria monocytogenes is well understood (1), much less is known regarding how Gram-negative pathogens, including the model professional intracytoplasmic Shigella species, enter the cytosol.During the course of an infection, many Gram-negative pathogens, including Shigella, Salmonella, enteropathogenic Escherichia coli, and Yersinia species, use type III secretion systems (T3SSs) as injection devices to deliver multiple virulence proteins, referred to as effectors, directly into the cytosol of infected cells (2). T3SSs are composed of ∼20 proteins and sense host cell contact via a tip complex at the distal end of a needle filament, which then acts as a scaffold for the formation of a translocon pore in the host cell membrane. Although components of their type III secretion apparatus (T3SA) are relatively well conserved, each pathogen delivers a unique repertoire of effectors into host cells, likely accounting for the establishment of a variety of replicative niches. For example, Salmonella and Shigella secreted effectors promote the uptake of these bacteria into nonphagocytic cells, whereas those from Yersinia inhibit phagocytosis by macrophages.All four pathogenic Shigella species—Shigella flexneri, Shigella sonnei, Shigella boydii, and Shigella dysenteriae—deliver ∼30 effectors into host cells, the majority of which are encoded on a large virulence plasmid (VP) alongside the genes for all of the proteins needed to form a T3SA (3). These secreted proteins play major roles in Shigella pathogenesis, including host cell invasion and modulation of innate immune response. One effector, IpgD, promotes the efficiency of Shigella phagosomal escape, although it is not absolutely required for this process (4). Interestingly, IpaB and IpaC, components of the Shigella translocon, the portion of the T3SA that inserts into the host cell membrane, have been implicated to mediate phagosomal escape based on the behavior of recombinant proteins (5–7). The physiologic relevance of these findings has not yet been directly addressed, as strains that lack either of these two proteins are completely impaired in the delivery of Shigella effectors into host cells (8).Here, using a reductionist approach, we directly tested a role for the Shigella translocon apparatus in phagosomal escape. Using an innovative reengineering approach, we introduced a functional effectorless Shigella T3SA into a nonpathogenic laboratory strain of DH10B E. coli. Remarkably, upon entry into host epithelial cells, these bacteria efficiently escape from phagosomes. This demonstrates for the first time, to our knowledge, in the context of an infection, a direct role for the Shigella T3SA in mediating vacuole lysis. Despite structural conservation across T3SS families, we further observed that, in the absence of any type III effectors, the Ysc T3SA mediates little to no Yersinia phagosomal escape, suggesting that not all injectisomes have equivalent functions. Lastly, by exploring the functional interchangeability of translocon components of the Shigella, Salmonella, and Chromobacterium T3SA, we demonstrate that one translocon protein controls the extent to which these intracellular pathogens escape into the cytosol of infected cells, thus demonstrating a major role for the T3SA in determining the site of the replicative niche of intracellular bacteria.     

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3′-5′-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.     

Ancient class of translocated oomycete effectors targets the host nucleus

Pathogens use specialized secretion systems and targeting signals to translocate effector proteins inside host cells, a process that is essential for promoting disease and parasitism. However, the amino acid sequences that determine host delivery of eukaryotic pathogen effectors remain mostly unknown. The Crinkler (CRN) proteins of oomycete plant pathogens, such as the Irish potato famine organism Phytophthora infestans, are modular proteins with predicted secretion signals and conserved N-terminal sequence motifs. Here, we provide direct evidence that CRN N termini mediate protein transport into plant cells. CRN host translocation requires a conserved motif that is present in all examined plant pathogenic oomycetes, including the phylogenetically divergent species Aphanomyces euteiches that does not form haustoria, specialized infection structures that have been implicated previously in delivery of effectors. Several distinct CRN C termini localized to plant nuclei and, in the case of CRN8, required nuclear accumulation to induce plant cell death. These results reveal a large family of ubiquitous oomycete effector proteins that target the host nucleus. Oomycetes appear to have acquired the ability to translocate effector proteins inside plant cells relatively early in their evolution and before the emergence of haustoria. Finally, this work further implicates the host nucleus as an important cellular compartment where the fate of plant–microbe interactions is determined.     

The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system

Type III secretion machines are essential for the biology of many bacteria that are pathogenic or symbiotic for animals, plants, or insects. They exert their function by delivering bacterial effector proteins into target eukaryotic cells. The core component of these machines is the needle complex, a multiprotein structure that spans the bacterial envelope and serves as a conduit for proteins that transit this secretion pathway. The needle complex is composed of a multiring base embedded in the bacterial envelope and a filament-like structure, the needle, that projects from the bacterial surface and is linked to the base by the inner rod. Assembly of the needle complex proceeds in a step-wise fashion that is initiated by the assembly of the base and is followed by the export of the building subunits for the needle and inner rod substructures. Once assembled, the needle complex reprograms its specificity and becomes competent for the secretion of effector proteins. Here through genetic, biochemical, and electron microscopy analyses of the Salmonella inner rod protein subunit PrgJ we present evidence that the assembly of the inner rod dictates the timing of substrate switching and needle length. Furthermore, the identification of mutations in PrgJ that specifically alter the hierarchy of protein secretion provides additional support for a complex role of the inner rod substructure in type III secretion.Type III secretion machines have evolved to shape the functional interface between many pathogenic or symbiotic bacteria with their respective hosts (1–3). These machines exert their function by delivering effector proteins into target cells with the capacity to modulate a variety of cellular processes for the benefit of the pathogens or symbionts that encode them (4). Type III secretion systems (TTSSs), comprising >20 proteins, are among the most complex protein secretion machines so far identified (5, 6). Salmonella Typhimurium, a cause of gastroenteritis in humans, encodes two TTSSs within its pathogenicity islands 1 (SPI-1) and 2 (SPI-2), which are essential for pathogenicity (7, 8). Through the delivery of a battery of ∼40 effector proteins, these machines allow S. Typhimurium to invade and survive within eukaryotic cells.TTSSs consist of the envelope-associated needle complex, which mediates the passage of the secreted proteins through the bacterial envelope, and several cytoplasmic accessory proteins, which are required for the recognition and sorting of the proteins destined to travel this pathway (5, 6, 9, 10). The needle complex is composed of a multiring, envelope-associated base substructure, and a filament or needle seated on the base, projecting outward from the bacterial surface (9, 11). The needle is linked to the base by the inner rod, an incompletely characterized substructure of the needle complex (12). Assembly of the needle complex is a coordinated process, which in the S. Typhimurium SPI-1-encoded TTSS is initiated by a subset of membrane proteins (SpaP, SpaQ, SpaR, and SpaS) that are thought to form a protein channel in the inner membrane (13, 14). The inner rings of the base assemble around these membrane proteins and after addition of the independently assembled outer rings, the base substructure becomes competent for type III secretion (13, 15). However, the base substructure has very narrow substrate specificity because it can only recognize the inner rod (PrgJ) and needle (PrgI) protein subunits and a regulatory protein (InvJ). Once the full needle complex is assembled, the type III secretion machine switches specificity to become competent for the secretion of effector proteins.The mechanisms by which the secretion machine is reprogramed are not completely understood although at least one regulatory protein, InvJ in the S. Typhimurium SPI-1 TTSS, is required for this process (16). In its absence, the needle complex is unable to switch substrates and consequently assembles abnormally long needles (16). The function of InvJ (or its homologs in other bacteria) is poorly understood and the subject of some controversy. For example, YscP, a functional homolog of InvJ in the pathogenic bacteria Yersinia enterocolitica, has been proposed to function as a molecular ruler, measuring the length of the needle and triggering substrate switching once an appropriate fixed length of the needle is achieved (17). How the measuring of the length would be carried out is not understood but it has been proposed that the fully extended form of YscP located within the lumen of the secretion channel measures the filament length by interacting with proteins at the tip and at the base of the needle complex, triggers substrate switching. An alternative model has been proposed in which InvJ is required for the assembly and/or linking of the inner rod to the base substructure, a process that leads to the firm anchoring of the needle filament to the base (12, 18). The anchoring of the needle filament leads to conformational changes on the cytoplasmic face of the needle complexes (11), which are thought to be essential for substrate switching. In this model the termination of the assembly of the inner rod is the crucial event that determines substrate switching. To better understand the role of the inner rod assembly in substrate switching we undertook biochemical, electron microscopy, and mutagenesis analyses of its building subunit, PrgJ. Collectively, our results support a role for the inner rod in determining the timing of substrate switching and needle length. Furthermore, the identification of mutations in the inner rod subunit that disrupt the hierarchy of protein secretion reveals an additional level of complexity in the function of this substructure in type III secretion.     

Role of the translocation partner in protection against AID-dependent chromosomal translocations

Chromosome translocations between Ig (Ig) and non-Ig genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is c-myc/IgH translocation, which is associated with Burkitt’s lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes. c-myc/IgH translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear c-myc/IgH translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCδ are required for protection against c-myc/IgH translocation, whereas Bcl-XL and BAFF enhance c-myc/IgH translocation. Whether these effects are general or specific to c-myc/IgH translocation and whether AID produces dsDNA breaks in genes other than c-myc and Ig is not known. To examine these questions, we developed an assay for translocation between IgH and Igβ, both of which are somatically mutated by AID. Igβ/IgH, like c-myc/IgH translocations, are AID-dependent, and AID is responsible for lesions on IgH and the non-IgH translocation partners. However, ATM, p53, and p19 do not protect against Igβ/IgH translocations. Instead, B cells are protected against Igβ/IgH translocations by a BAFF- and PKCδ-dependent pathway. We conclude that AID-induced double-strand breaks in non-Ig genes other than c-myc lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells.     

The first deletion mutation in the TSP1-6 repeat domain of ADAMTS13 in a family with inherited thrombotic thrombocytopenic purpura

The inherited deficiency of ADAMTS13 is usually associated with severe forms of thrombotic thrombocytopenic purpura. Among the mutations identified in the ADAMTS13 gene, none have been described on the TSP1-6 repeat domain. We investigated an Iranian family with a history of chronic recurrent thrombotic thrombocytopenic purpura, severe ADAMTS13 deficiency and a heterogeneous pattern of clinical symptoms among affected members. Genetic analysis revealed a homozygous deletion of nucleotides 2930–2935 (GTGCCC) in exon 23 of ADAMTS13, leading to the replacement of Cys977 by a Trp and the deletion of Ala978 and Arg979 in the TSP1-6 repeat domain. To explore the mechanism of ADAMTS13 deficiency, in vitro expression studies were performed. Western blotting, pulse-chase labeling and immunofluorescence studies demonstrated a secretion pathway defect of the mutant protein, with no intracellular accumulation. This finding is consistent with the severe ADAMTS13 deficiency but does not explain the heterogeneous clinical picture of the 3 siblings carrying the same mutation.     

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.     

Clostridium ljungdahlii represents a microbial production platform based on syngas

Clostridium ljungdahlii is an anaerobic homoacetogen, able to ferment sugars, other organic compounds, or CO₂/H₂ and synthesis gas (CO/H₂). The latter feature makes it an interesting microbe for the biotech industry, as important bulk chemicals and proteins can be produced at the expense of CO₂, thus combining industrial needs with sustained reduction of CO and CO₂ in the atmosphere. Sequencing the complete genome of C. ljungdahlii revealed that it comprises 4,630,065 bp and is one of the largest clostridial genomes known to date. Experimental data and in silico comparisons revealed a third mode of anaerobic homoacetogenic metabolism. Unlike other organisms such as Moorella thermoacetica or Acetobacterium woodii, neither cytochromes nor sodium ions are involved in energy generation. Instead, an Rnf system is present, by which proton translocation can be performed. An electroporation procedure has been developed to transform the organism with plasmids bearing heterologous genes for butanol production. Successful expression of these genes could be demonstrated, leading to formation of the biofuel. Thus, C. ljungdahlii can be used as a unique microbial production platform based on synthesis gas and carbon dioxide/hydrogen mixtures.     

O-Glycosylation regulates polarized secretion by modulating Tango1 stability

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. Here, we demonstrate that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. Our studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, our data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.Regulation of secretory vesicle formation and polarized secretion in vivo is crucial to ensure the proper deposition of signaling molecules, morphogens, and matrix components that mediate growth and differentiation. Polarized secretion is also required in many differentiated tissues, such as the digestive tract, where secreted components along the apical surface form the mucous membrane that confers protection from mechanical and microbial insults (1) and provides immunoregulatory signals (2). Indeed, disruptions in the secreted mucous membrane are associated with diseases of the digestive tract, such as colitis and colon cancer (3–6).Recent studies aimed at identifying the factors that influence secretion have elucidated novel proteins that function in unique aspects of secretion, including the enzymes responsible for the addition of sugars to mucins and other proteins (mucin-type O-glycosylation) (7). O-glycosylation is an essential, evolutionarily conserved protein modification (8, 9) that has direct medical relevance, as aberrations are responsible for the human diseases familial tumoral calcinosis (10, 11) and Tn syndrome (12). Loss of this protein modification affected constitutive secretion and Golgi apparatus structure in Drosophila cell culture (7, 13) and secretion in the developing respiratory system in vivo (14). Additionally, loss of O-glycosylation also disrupted secretion of an extracellular matrix protein (Tiggrin) in the developing wing, resulting in aberrant basement membrane formation and disrupted integrin-mediated cell adhesion (15). Mammalian studies have confirmed the effects of O-glycosylation on secretion, as loss of a glycosyltransferase (ppGalNAcT-1) disrupted secretion of laminin and collagen during mammalian organogenesis, altering the composition of the basement membrane and disrupting proper FGF signaling and organ growth (16). Although these studies all point to a role for O-glycosylation in secretion, the mechanisms by which this protein modification affects secretion are currently unknown. Here, we demonstrate that O-glycosylation influences secretion and secretory vesicle formation by glycosylating the essential endoplasmic reticulum (ER)/Golgi protein Tango1, and conferring protection from furin-mediated proteolysis. Interestingly, secretory defects caused by the loss of O-glycosylation could be rescued by reducing the activity of a specific furin (Dfur2) in vivo. Our results elucidate a novel regulatory paradigm whereby the competing activities of an O-glycosyltransferase and a furin control secretion and secretory vesicle formation. Moreover, this finding offers a potential treatment for disorders of glycosylation by modulating the activity of specific proteases in vivo.     

Independent gene duplications of the YidC/Oxa/Alb3 family enabled a specialized cotranslational function

YidC/Oxa/Alb3 family proteins catalyze the insertion of integral membrane proteins in bacteria, mitochondria, and chloroplasts, respectively. Unlike gram-negative organisms, gram-positive bacteria express 2 paralogs of this family, YidC1/SpoIIIJ and YidC2/YgjG. In Streptococcus mutans, deletion of yidC2 results in a stress-sensitive phenotype similar to that of mutants lacking the signal recognition particle (SRP) protein translocation pathway, while deletion of yidC1 has a less severe phenotype. In contrast to eukaryotes and gram-negative bacteria, SRP-deficient mutants are viable in S. mutans; however, double SRP-yidC2 mutants are severely compromised. Thus, YidC2 may enable loss of the SRP by playing an independent but overlapping role in cotranslational protein insertion into the membrane. This is reminiscent of the situation in mitochondria that lack an SRP pathway and where Oxa1 facilitates cotranslational membrane protein insertion by binding directly to translation-active ribosomes. Here, we show that OXA1 complements a lack of yidC2 in S. mutans. YidC2 also functions reciprocally in oxa1-deficient Saccharomyces cerevisiae mutants and mediates the cotranslational insertion of mitochondrial translation products into the inner membrane. YidC2, like Oxa1, contains a positively charged C-terminal extension and associates with translating ribosomes. Our results are consistent with a gene-duplication event in gram-positive bacteria that enabled the specialization of a YidC isoform that mediates cotranslational activity independent of an SRP pathway.     

Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa

The siderophore pyoverdine (PVD) is a primary virulence factor of the human pathogenic bacterium Pseudomonas aeruginosa, acting as both an iron carrier and a virulence-related signal molecule. By exploring a number of P. aeruginosa candidate systems for PVD secretion, we identified a tripartite ATP-binding cassette efflux transporter, here named PvdRT-OpmQ, which translocates PVD from the periplasmic space to the extracellular milieu. We show this system to be responsible for recycling of PVD upon internalization by the cognate outer-membrane receptor FpvA, thus making PVD virtually available for new cycles of iron uptake. Our data exclude the involvement of PvdRT-OpmQ in secretion of de novo synthesized PVD, indicating alternative pathways for PVD export and recycling. The PvdRT-OpmQ transporter is one of the few secretion systems for which substrate recognition and extrusion occur in the periplasm. Homologs of the PvdRT-OpmQ system are present in genomes of all fluorescent pseudomonads sequenced so far, suggesting that PVD recycling represents a general energy-saving strategy adopted by natural Pseudomonas populations.     

Association of Periodontal Diseases and Liver Fibrosis in Patients With HCV and/or HBV infection

Background:

Periodontal disease and systemic health are closely associated. However, there is no data supporting the association between periodontal disease and patients with liver diseases associated with hepatitis C virus (HCV) and/or hepatitis B virus (HBV) infection.

Objectives:

The aim of this study was to evaluate the association between periodontitis and progression of liver diseases in patients with HCV and/or HBV infection.

Patients and Methods:

In this retrospective study, 351 patients with HCV- and/or HBV-related liver diseases underwent screening for periodontal disease using the Salivaster® salivary occult blood test from February 2010 to June 2014. Furthermore, we examined the prevalence of fimbrillin (fimA) genotype of Porphyromonas gingivalis (P. gingivalis) in 28 HCV-infected patients visited at our hospital between January 2013 and June 2014. P. gingivalis with fimA genotype with types I to V was further detected using a PCR method.

Results:

Of 351 patients, 76 patients (group 1) had a strong positive result for salivary occult blood test and 275 patients (group 2) had weak positive or negative test results. Significant factors between the groups were obesity, level of AST, ALT, LDH, ALP, Alb, D.Bil, T.cho, AFP, platelets (Plt), IRI, HOMA-IR, current interferon (IFN) treatment and the daily frequency of tooth brushing. Between-groups analysis indicated that total protein (T.pro) level and liver fibrosis were significant factors. According to multivariate analysis, five factors were associated with periodontal disease as Plt count below 80000, brushing teeth only once a day, current IFN treatment, aged 65 years or older and obesity. The adjusted odds ratios for these five factors were 5.80, 3.46, 2.87, 2.50 and 2.33, respectively, and each was statistically significant. Twenty-eight saliva specimens had positive results for P. gingivalis with fimA genotype types I to V. The prevalence of fimA genotype II was higher in 14 patients with liver cirrhosis or a history of hepatocellular carcinoma treatment (group B, 50.00%) than 14 patients with only hepatitis C (group A, 21.43%).

Conclusions:

Periodontitis might be associated with progression of viral liver disease; hence, controlling oral disease is essential for the prevention and management of liver fibrosis.     

The periodontal pathogen Porphyromonas gingivalis cleaves apoB-100 and increases the expression of apoM in LDL in whole blood leading to cell proliferation

Objective. Several studies support an association between periodontal disease and atherosclerosis with a crucial role for the pathogen Porphyromonas gingivalis. This study aims at investigating the proteolytic and oxidative activity of P. gingivalis on LDL in a whole blood system using a proteomic approach and analysing the effects of P. gingivalis‐modified LDL on cell proliferation. Methods. The cellular effects of P. gingivalis in human whole blood were assessed using lumi‐aggregometry analysing reactive oxygen species production and aggregation. Blood was incubated for 30 min with P. gingivalis, whereafter LDL was isolated and a proteomic approach was applied to examine protein expression. LDL‐oxidation was determined by analysing the formation of protein carbonyls. The effects of P. gingivalis‐modified LDL on fibroblast proliferation were studied using the MTS assay. Results. Incubation of whole blood with P. gingivalis caused an extensive aggregation and ROS production, indicating platelet and leucocyte activation. LDL prepared from bacteria‐exposed blood showed an increased protein oxidation, elevated levels of apoM and formation of two apoB‐100 N‐terminal fragments. Porphyromonas gingivalis‐modified LDL markedly increased the growth of fibroblasts. Inhibition of gingipain R suppressed the modification of LDL by P. gingivalis. Conclusions. The ability of P. gingivalis to change the protein expression and proliferative capacity of LDL may represent a crucial event in periodontitis‐associated atherosclerosis.     

Correlation between group behavior and quorum sensing in Pseudomonas aeruginosa isolated from patients with hospital-acquired pneumonia

Background

This study investigated the correlation between the expression of the Las and Rhl quorum-sensing (QS) systems and the communal behavior (motility, biofilm formation, and pyocyanin production) of Pseudomonas aeruginosa (P. aeruginosa) isolated from patients with hospital-acquired pneumonia.

Methods

We analyzed 138 P. aeruginosa isolates from 48 patients (30 men and 18 women; age 68.18±15.08 years). P. aeruginosa clinical isolates were assessed for Las and Rhl gene expression and bacterial motility, biofilm formation, and pyocyanin production.

Results

P. aeruginosa swimming, twitching, and swarming motility positively correlated with the expression of LasI, LasR, and RhlI (P<0.05) but not with that of RhlR (P>0.05). At all analyzed time points, a significant positive correlation was found between biofilm formation and the expression of LasI, LasR (P<0.01), and RhlI (P<0.05 for day 1, P<0.01 for days 7 and 14), whereas RhlR expression positively correlated with biofilm formation only on day 14 (P<0.05). On days 1 and 7, positive correlation was observed between pyocyanin production and the levels of LasI and RhlI (P<0.05). In bacterial clearance cases, the expression of QS-related genes and the group behavior of the pathogen did not correlate (P>0.05). However, in cases of persistent P. aeruginosa infection, the changes in LasI and LasR gene expression were positively correlated with those in bacterial motility (P<0.05), and the changes in LasI, LasR, RhlI, and RhlR expression showed a significant positive association with those in biofilm formation (P<0.01).

Conclusions

In patients with hospital-acquired pneumonia, the expression of the Las and Rhl QS genes was associated with bacterial motility, biofilm formation, and pyocyanin production, suggesting an involvement of the QS genes in the clearance of pathogenic P. aeruginosa in patients.     

Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of nonmotile mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nonmotile mutant of F. johnsoniae (UW102-09) with a library of wild-type DNA by using the shuttle cosmid pCP17. The complementing plasmid (pCP100) contained an insert of 13 kbp, and restored motility to 4 of 61 independently isolated nonmotile mutants. A 1.3-kbp fragment that encompassed a single ORF, gldA, complemented all four mutants. Disruption of the chromosomal copy of gldA in wild-type F. johnsoniae UW101 eliminated gliding motility. The predicted protein produced by gldA has strong sequence similarity to ATP binding cassette transport proteins.     

Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.The Gram-negative intracellular bacterium Legionella pneumophila is an opportunistic human pathogen responsible for Legionnaires’ disease. The bacteria are naturally found in freshwater systems where they replicate within protozoan hosts (1). It is thought that the adaptation to replication within amoebas has equipped L. pneumophila with the factors required to replicate successfully within human macrophages following opportunistic infection (2). Through genome sequencing, we have discovered that L. pneumophila encodes a high number and variety of proteins similar in sequence to eukaryotic proteins that are never or rarely found in other prokaryotic genomes (3). Subsequent phylogenetic analyses have suggested that many of these proteins were acquired by horizontal gene transfer (3, 4). One of these proteins exhibits a high degree of similarity to eukaryotic sphingosine-1 phosphate lyase (SPL). The L. pneumophila SPL homolog (LpSpl encoded by gene lpp2128, lpg2176, or legS2) is conserved in all L. pneumophila strains sequenced to date, but absent from Legionella longbeachae (SI Appendix, Table S1). Phylogenetic analysis of SPL sequences showed that the L. pneumophila spl gene was most likely acquired early during evolution by horizontal gene transfer from a protozoan organism (4, 5). With the increase in genome sequences available, SPL homologs have now been identified in other bacteria such as Roseiflexus, Myxococcus, Stigmatella, and Symbiobacterium (6).Eukaryotic SPL tightly regulates intracellular levels of sphingosine-1-phosphate (S1P). Sphingolipids are ubiquitous building blocks of eukaryotic cell membranes, and the sphingolipid metabolites ceramide, ceramide-1-phosphate, sphingosine, and S1P are key signaling molecules that regulate many cellular processes important in immunity, inflammation, infection, and cancer (7). SPL uses pyridoxal 5′-phosphate (PLP) as a cofactor to irreversibly degrade S1P into phosphoethanolamine and hexadecenal (SI Appendix, Fig. S1). Structural analysis of SPL from Symbiobacterium thermophilum (StSPL) and Saccharomyces cerevisiae (Dpl1p) identified the residues involved in activity and proposed a mechanism for S1P cleavage (8). Structural elucidation of human SPL (hSPL) showed that the yeast and the human enzymes adopt largely the same structures (9).Recent work suggests a possible link between the role of lipids in the regulation of apoptosis and autophagy (10). Autophagy is an evolutionary conserved pathway controlling the quality and quantity of eukaryotic organelles and the cytoplasmic biomass. Double-membrane vesicles called “autophagosomes” engulf nonfunctional or damaged cellular components and deliver them to lysosomes, where the content is degraded (11). Furthermore, it has been shown that autophagy acts as a cell-autonomous defense mechanism against intracellular bacteria, contributing to antibacterial immunity by regulating the inflammatory immune response and routing engulfed intracellular bacteria toward lysosomal degradation (12, 13). Many pathogens are able to evade autophagy, although the molecular mechanisms at play remain largely uncharacterized (14–20). Among these pathogens L. pneumophila is known to escape cellular attack by blocking autophagy defenses (21). Although it has been reported that L. pneumophila interferes with the autophagy machinery and with host factors that play a role in the cellular defense (22), only two L. pneumophila proteins that target the autophagy machinery, RavZ and LegA9, have been identified (23, 24). The bacterial effector RavZ is a cysteine protease that cleaves and causes delipidation of the autophagosome protein LC3, thereby dampening the autophagy process (23, 25). Interestingly, RavZ is not present in all strains of L. pneumophila, but in all strains tested, L. pneumophila-mediated disruption of autophagosomal maturation delays and/or prevents the Legionella-containing vacuole (LCV) from fusing with lysosomes (26). Thus, L. pneumophila likely also employs other mechanisms to restrain autophagy, and LpSpl, present in all strains sequenced to date, is a good candidate. A first characterization of LpSpl in strain JR32 showed that this protein is secreted by the Dot/Icm type IV secretion system and that it complements the sphingosine-sensitive phenotype of a S. cerevisiae SPL-null mutant, suggesting that it indeed has SPL activity. However, no functional analyses were reported (5). Thus, we aimed to understand the function of this SPL homolog in L. pneumophila.Here we report the crystal structure of LpSpl, identify the active site, and show that LpSpl indeed confers S1P lyase activity to L. pneumophila. Its activity during infection leads to the specific reduction of cellular levels of sphingosine, whereas other sphingolipids are down-regulated in a LpSpl-independent manner. Furthermore, we confirm that LpSpl plays a role in delaying the autophagy response of the cell during infection and is important for infection in a pulmonary mouse model of Legionnaires’ disease.     

Molecular ruler determines needle length for the Salmonella Spi-1 injectisome

The type-III secretion (T3S) systems of bacteria are part of self-assembling nanomachines: the bacterial flagellum that enables cells to propel themselves through liquid and across hydrated surfaces, and the injectisome that delivers pathogenic effector proteins into eukaryotic host cells. Although the flagellum and injectisome serve different purposes, they are evolutionarily related and share many structural similarities. Core features to these T3S systems are intrinsic length control mechanisms for external cellular projections: the hook of the flagellum and the injectisome needle. We present evidence that the Spi-1 injectisome, like the Salmonella flagellar hook, uses a secreted molecular ruler, InvJ, to determine needle length. This result supports a universal length control mechanism using molecular rulers for T3S systems.Length control mechanisms of macromolecular structures are used throughout biology for maintaining cellular functions (1–6). Mechanisms of length control have evolved independently and range from the kinetically controlled length of the metaphase spindle involved in eukaryotic cell-division, to the scaffolds controlling bacteriophage tail-lengths (2, 5). One of the best-studied and controversial mechanisms of length control is that of bacterial Type III secretion (T3S) system, which includes the virulence-associated injectisome of Salmonella pathogenicity island 1 (Spi-1), and the bacterial motility organelle, the flagellum.The injectisome is a hypodermic needle-like organelle used by pathogenic Gram-negative bacteria to inject effector proteins into eukaryotic host cells (7). This complex organelle is embedded in the membranes and cell wall of Gram-negative bacteria. The basal structure of the injectisome includes a rod-like component that spans the periplasmic space between the inner and outer membranes, and an injectisome-specific T3S apparatus within the cytoplasmic membrane (7–9). The T3S system associated with the injectisome secretes proteins required for its structure and assembly, as well as effector proteins into host cells to facilitate pathogenesis. Extending from the injectisome basal-body at the cell surface is a rigid needle-like structure, which grows to defined lengths for different bacterial species, and serves as a conduit between the bacterium and the host cell (10–12). At the tip of the needle is the translocon, which allows the needle to dock to the host membrane (13–15) (Fig. 1A).Open in a separate windowFig. 1.(A) Schematic of axial components in a cross-section of the Spi-1 injectisome. (B) Schematic of axial components of the bacterial flagellum. (C) Model of hook-length determination by the infrequent ruler mechanism: (i) The secreted N terminus of FliK (FliK_N), shown in red halts hook polymerization and is slow to diffuse. (ii) The lack of interactions in short hooks or the folding of the secreted FliK_N as it exits the secretion channel rapidly pulls the FliK molecule past FlhB (shown in orange) through the channel without induction of the secretion-specificity switch. (iii) FliK is secreted outside of the cell and hook polymerization continues. (iv) The hook has grown to the physiological length and a new FliK molecule is secreted, again halting hook growth. (v) The C terminus of FliK (FliK_C) is now closely aligned to FlhB, the slower rate of FliK secretion provides sufficient time for a productive FliK–FlhB interaction that induces the secretion-specificity switch. (D) The static ruler model for length control of the Yersinia injectisome: (i) A single YscP (shown in red) molecule enters the secretion channel and remains there during needle polymerization. The N terminus of YscP interacts with the needle or tip-complex and is pulled through as the needle continues to polymerize, bringing the C terminus of YscP (YscP_C) near the secretion apparatus (YscU – shown in orange). (ii) When YscP_C and YscU are close enough to interact, a secretion specificity switch occurs, halting needle polymerization. (E) The inner-rod model of length control of the Spi-1 injectisome: Here, both the inner-rod (shown in green) and needle (shown in blue) are assembled simultaneously. (i) Assembly of the inner-rod is facilitated by InvJ (shown in red). (ii) The completion of the inner-rod leads to substrate switching and the interruption of secretion of the inner-rod and needle proteins, thus determining the length of the needle substructure.Like the injectisome, the flagellum contains a membrane and cell wall spanning basal-body, which also functions as a rotary motor in motility, and includes an integral membrane-associated, flagellar-specific T3S apparatus (16, 17) (Fig. 1B). From the basal-body at the cell surface extends a flexible tube, called the hook, which also grows to defined lengths and serves as a universal-joint to transmit torque generated by the basal-body to a long (up to 20 µm) helical propeller-like structure called the flagellar filament (18, 19). A defining characteristic of T3S systems is an intrinsic length control mechanism for hook and needle length that is associated with a switch in the type of substrates that are recognized and exported by the secretion apparatus (3, 4, 20, 21). For the flagellum of Salmonella Typhimurium, assembly of the basal-body is followed by the secretion of ∼130 FlgE protein subunits that polymerize to form the hook (19). When the hook reaches a terminal length of ≥ 40 nm, the T3S apparatus switches secretion specificity to filament or late-type secretion substrates to complete flagellum assembly (20, 22). Here, the length of the hook and induction of the secretion specificity switch is determined by a secreted, molecular ruler called FliK (23–25).Assembly of the injectisome proceeds in a similar fashion to the flagellum; however, the ring structures that are in the outer membrane form independent to the inner membrane-associated components of the basal-body (10, 26, 27). Subunits that make up an inner-rod structure that spans the cell wall and periplasmic space and subunits that assembly into the external needle are part of the early class of secreted substrates (8, 12). The injectisome is completed when the needle reaches an optimal length after which, the T3S system switches specificity for late secreted substrates including the translocon and effector proteins (10, 21, 28).Many mechanisms have been proposed for length determination for the hook of the flagellar system and the needle of the injectisome system. Length control of the flagellar hook and injectisome needle by a molecular ruler was first proposed based on work on the Yersinia spp. injectisome system (3). For the injectisome system of Yersinia enterocolitica, the needles grow to a length of 58 ± 10 nm, whereas Yersinia pestis needles grow to a length of about 41 nm (3). It was shown that the N-terminal length of YscP, a nonstructural, early secretion substrate and functional homolog of FliK, determined needle lengths of the Yersinia injectisomes (10, 29, 30). An important difference between the YscP molecules from Y. enterocolitica and Y. pestis is that Y. pestis YscP is 60 amino acid residues shorter in length, which corresponded to shorter needles. Increasing or decreasing the lengths of the N-terminal domain of YscP or FliK resulted in a linear correlation between the length of the needle and the length of YscP, or the length of the hook and FliK (3). Additionally, the C-terminal domain of YscP and FliK is believed to interact with the component of the T3S apparatus that governs secretion specificity, known as YscU in Yersinia spp. and FlhB in the flagellar system. Loss of YscP/FliK, or dominant mutant alleles in the T3S switch component yscU/flhB, will give rise to a poly-needle and poly-hook phenotype, respectively, where the needles or hooks polymerize indefinitely and do not switch secretion specificity modes (21, 31).Although both the flagellum of S. Typhimurium and injectisome of Yersinia spp. use molecular rulers for controlling length, the mechanisms for how measurements are achieved are proposed to work in different ways (22, 28, 32). In Yersina injectisomes, a static-ruler model has been proposed (32). Here, the ruler molecule remains anchored by its N terminus to the tip of the needle in the secretion channel as the needle substrates are secreted and polymerize into the growing needle. The anchored YscP is pulled along with the growing needle until its C terminus is in proximity to the secretion apparatus to induce the secretion specificity switch (Fig. 1D). Alternatively, in the flagellar system, FliK has been shown to act as an infrequent ruler, with multiple FliK molecules secreted through the growing structure until a minimal hook-length is achieved (22, 33). In this model, the probability of inducing the secretion specificity switch is a function of the velocity of FliK secretion, which is inversely correlated to hook length. FliK enters the secretion channel and proceeds to the hook-tip and exits the cell from its N terminus (FliK_N) followed by the C terminus (FliK_C). Once FliK_N exits the cell, FliK_C continues through the channel at an increased rate of secretion that is too fast to allow FliK_C to interact with FlhB (22). It is only when the hook has reached a sufficient length where FliK_C can interact with FlhB prior to the exit of FliK_N from the cell does the secretion specificity switch at FlhB occur (Fig. 1C). Interestingly, injectisomes in S. enterica were proposed to measure needle lengths by a mechanism that is different from those proposed for FliK and YscP.S. enterica encodes two injectisomes: the Salmonella pathogenicity island 1 (Spi-1) and Salmonella pathogenicity island 2 (Spi-2) injectisomes, which are required to cause gasteroenteritis in humans (34). The Spi-1 needle is composed of PrgI subunits that polymerize to a length of ∼25 nm before undergoing the secretion specificity switch. An early secretion substrate, InvJ, was believed to be the molecular ruler analog in Spi-1 injectisomes, because its absence results in a poly-needle phenotype; however, these injectisomes were reported to be missing their inner-rod structure (10, 28). Additionally, overexpression of the inner-rod subunit protein PrgJ was found to result in slightly shorter needle lengths, giving rise to an inner-rod length-control model for the Spi-1 needle (28). In this model, needle length is determined by the rate of inner-rod assembly, where the complete inner-rod structure presumably interacts with the T3S apparatus to induce the secretion specificity switch, thus terminating needle assembly (Fig. 1E). A recent study supporting the inner-rod model reports the isolation of mutant PrgJ-alleles that give rise to abnormally long needles that still undergo the secretion specificity switch and assemble the inner-rod (35). It is reasoned that these mutant alleles are defective in PrgJ–PrgJ interactions causing slower inner-rod assembly, and therefore longer needles. Still, it remained unclear as to what role InvJ plays in Spi-1 injectisome needle length control. In this study, we address the hypothesis that InvJ, like its functional homologs FliK and YscP, acts as a molecular ruler in determining needle length for the Spi-1 injectisome. Our work demonstrates that InvJ acts as a molecular ruler and supports a universal molecular ruler model for length control in T3S systems.