The Sde phosphoribosyl-linked ubiquitin transferases protect the Legionella pneumophila vacuole from degradation by the host.

Legionella pneumophila grows intracellularly within the membrane-bound Legionella-containing vacuole (LCV) established by proteins translocated via the bacterial type IV secretion system (T4SS). The Sde family, one such group of translocated proteins, catalyzes phosphoribosyl-ubiquitin (pR-Ub) modification of target substrates. Mutational loss of the entire Sde family results in small defects in intracellular growth, making it difficult to identify a clear role for this posttranslational modification in supporting the intracellular lifestyle. Therefore, mutations that aggravate the loss of sde genes and caused intracellular growth defects were identified, providing a mechanistic connection between Sde function and vacuole biogenesis. These double mutants drove the formation of LCVs that showed vacuole disintegration within 2 h of bacterial contact. Sde proteins appeared critical for blocking access of membrane-disruptive early endosomal membrane material to the vacuole, as RNAi depletion of endosomal pathway components partially restored LCV integrity. The role of Sde proteins in preventing host degradation of the LCV was limited to the earliest stages of infection. The time that Sde proteins could prevent vacuole disruption, however, was extended by deletion of sidJ, which encodes a translocated protein that inactivates Sde protein active sites. These results indicate that Sde proteins act as temporally regulated vacuole guards during the establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments during the earliest steps of LCV biogenesis.

Deciphering the Impact of Defecation Frequency on Gut Microbiome Composition and Diversity.

This study explores the impact of defecation frequency on the gut microbiome structure by analyzing fecal samples from individuals categorized by defecation frequency: infrequent (1-3 times/week, n = 4), mid-frequent (4-6 times/week, n = 7), and frequent (daily, n = 9). Utilizing 16S rRNA gene-based sequencing and LC-MS/MS metabolome profiling, significant differences in microbial diversity and community structures among the groups were observed. The infrequent group showed higher microbial diversity, with community structures significantly varying with defecation frequency, a pattern consistent across all sampling time points. The Ruminococcus genus was predominant in the infrequent group, but decreased with more frequent defecation, while the Bacteroides genus was more common in the frequent group, decreasing as defecation frequency lessened. The infrequent group demonstrated enriched biosynthesis genes for aromatic amino acids and branched-chain amino acids (BCAAs), in contrast to the frequent group, which had a higher prevalence of genes for BCAA catabolism. Metabolome analysis revealed higher levels of metabolites derived from aromatic amino acids and BCAA metabolism in the infrequent group, and lower levels of BCAA-derived metabolites in the frequent group, consistent with their predicted metagenomic functions. These findings underscore the importance of considering stool consistency/frequency in understanding the factors influencing the gut microbiome.

Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage.

Hfq plays important roles in virulence and stress adaptation in Cronobacter sakazakii ATCC 29544.

Cronobacter spp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated with Cronobacter infection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq in C. sakazakii virulence. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion (3-fold) into animal cells and survival (10(3)-fold) within host cells, and exhibited low resistance to hydrogen peroxide (10(2)-fold). Remarkably, the loss of hfq led to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes.

Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Promote Establishment and Maintenance of the Legionella Replication Vacuole.

The Legionella pneumophila Sde family of translocated proteins promotes host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity was tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, prevented binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb resulted in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decayed quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.

The Sde Phosphoribosyl-Linked Ubiquitin Transferases protect the Legionella pneumophila vacuole from degradation by the host.

Legionella pneumophila grows intracellularly within a host membrane-bound vacuole that is formed in response to a bacterial type IV secretion system (T4SS). T4SS translocated Sde proteins promote phosphoribosyl-linked ubiquitination of endoplasmic reticulum protein Rtn4, but the role played by this modification is obscure due to lack of clear growth defects of mutants. To identify the steps in vacuole biogenesis promoted by these proteins, mutations were identified that unmasked growth defects in Δ sde strains. Mutations in the sdhA , ridL and legA3 genes aggravated the Δ sde fitness defect, resulting in disruption of the Legionella -containing vacuole (LCV) membrane within 2 hrs of bacterial contact with host cells. Depletion of Rab5B and sorting nexin 1 partially bypassed loss of Sde proteins, consistent with Sde blocking early endosome and retrograde trafficking, similar to roles previously demonstrated for SdhA and RidL proteins. Sde protein protection of LCV lysis was only observed shortly after infection, presumably because Sde proteins are inactivated by the metaeffector SidJ during the course of infection. Deletion of SidJ extended the time that Sde proteins could prevent vacuole disruption, indicating that Sde proteins are negatively regulated at the posttranslational level and are limited to protecting membrane integrity at the earliest stages of replication. Transcriptional analysis was consistent with this timing model for an early point of execution of Sde protein. Therefore, Sde proteins act as temporally-regulated vacuole guards during establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments early during biogenesis of the LCV. Significance statement: Maintaining replication compartment integrity is critical for growth of intravacuolar pathogens within host cells. By identifying genetically redundant pathways, Legionella pneumophila Sde proteins that promote phosphoribosyl-linked ubiquitination of target eukaryotic proteins are shown to be temporally-regulated vacuole guards, preventing replication vacuole dissolution during early stages of infection. As targeting of reticulon 4 by these proteins leads to tubular endoplasmic reticulum aggregation, Sde proteins are likely to construct a barrier that blocks access of disruptive early endosomal compartments to the replication vacuole. Our study provides a new framework for how vacuole guards function to support biogenesis of the L. pneumophila replicative niche.

Identification of Genes Required for Long-Term Survival of Legionella pneumophila in Water.

Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for facilitating epidemic outbreaks. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42°C. The mutants with the most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress. In addition, periplasmically localized proteins that are known (EnhC) or hypothesized (lpg1697) to stabilize the cell wall against turnover were essential for water survival. To test that the identified mutations disrupted water survival, candidate genes were knocked down by CRISPRi. The vast majority of knockdown strains with verified transcript depletion showed remarkably low viability after 50-day incubations. To demonstrate that maintenance of cell wall integrity was an important survival determinant, a deletion mutation in lpg1697, in a gene encoding a predicted l,d-transpeptidase domain, was analyzed. The loss of this gene resulted in increased osmolar sensitivity and carbenicillin hypersensitivity relative to the wild type, as predicted for loss of an l,d-transpeptidase. These results indicate that the L. pneumophila envelope has been evolutionarily selected to allow survival under conditions in which the bacteria are subjected to long-term exposure to starvation and low osmolar conditions. IMPORTANCE Water is the primary vector for transmission of L. pneumophila to humans, and the pathogen is adapted to persist in this environment for extended periods of time. Preventing survival of L. pneumophila in water is therefore critical for prevention of Legionnaires' disease. We analyzed dense transposon mutation pools for strains with severe survival defects during a 50-day water incubation at 42°C. By tracking the associated transposon insertion sites in the genome, we defined a distinct essential gene set for water survival and demonstrate that a predicted peptidoglycan cross-linking enzyme, lpg1697, and components of the electron transport chain are required to ensure survival of the pathogen. Our results indicate that select characteristics of the cell wall and components of the respiratory chain of L. pneumophila are primary evolutionary targets being shaped to promote its survival in water.

Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Promote Establishment and Maintenance of the Legionella Replication Vacuole.

The Legionella pneumophilaSde family of translocated proteins promote host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity was tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, caused an absolute block in binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb resulted in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decayed quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.

SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase.

Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.

The interaction between ubiquitin and yeast polymerase η C terminus does not require the UBZ domain.

Polymerase η (Polη) is one of the Y-family polymerases that is recruited by monoubiquitinated proliferating cell nuclear antigen (Ub-PCNA) to DNA damage sites during translesion synthesis (TLS). This interaction is mediated by an ubiquitin-binding zinc-finger (UBZ) domain and a PCNA-interacting protein (PIP) box in Polη, which binds to ubiquitin and PCNA, respectively. Here, we show that without the UBZ domain, the PIP box of yeast Polη has a novel binding function with ubiquitin. Furthermore, the UBZ domain and the PIP box share the same binding surfaces for ubiquitin. The interaction with ubiquitin via the PIP box stabilizes the Ub-PCNA/Polη complex. Moreover, the PIP residues I624 and L625 contribute to Polη function in TLS in vivo.

Corrigendum: Secretion of Salmonella Pathogenicity Island 1-Encoded Type III Secretion System Effectors by Outer Membrane Vesicles in Salmonella Enterica Serovar Typhimurium.

[This corrects the article DOI: 10.3389/fmicb.2018.02810.].

New virulence factor CSK29544_02616 as LpxA binding partner in Cronobacter sakazakii.

Cronobacter sakazakii is an opportunistic pathogen that can cause meningitis and necrotizing enterocolitis in premature infants, but its virulence determinants remain largely unknown. In this study, a transposon-mediated random-mutant library of C. sakazakii was used to identify new virulence factors. Compared to wild-type bacteria, a mutant lacking CSK29544_02616 (referred to as labp) was defective in invasion into intestinal epithelial cells (by at least 1000-fold) and showed less phagocytosis by macrophages (by at least 50-fold). The lack of labp in C. sakazakii changed the profile of outer membrane proteins, decreased the production of lipopolysaccharides, and increased the production of membrane phospholipids. Bacterial physiological characteristics including surface hydrophobicity and motility were also altered in the absence of labp, presumably because of changes in the bacterial-envelope structure. To systematically determine the role of labp, ligand fishing was conducted using Labp as a bait, which revealed LpxA as a binding partner of Labp. LpxA is UDP-N-acetylglucosamine (GlcNAc) acyltransferase, the first enzyme in the pathway of lipid A biosynthesis. Labp increased the enzymatic activity of LpxA without influencing lpxA expression. Considering multifaceted roles of lipopolysaccharides in virulence regulation, Labp is a novel virulence factor that promotes the production of lipid A by LpxA in Cronobacter.

Secretion of Salmonella Pathogenicity Island 1-Encoded Type III Secretion System Effectors by Outer Membrane Vesicles in Salmonella enterica Serovar Typhimurium.

Outer membrane vesicles (OMVs) are spherical membranous structures released by Gram-negative bacteria. Several bacterial pathogens utilize OMVs as vehicles for the delivery of virulence factors into host cells. Results of our previous study on proteomic analysis revealed that OMVs isolated from Salmonella enterica serovar Typhimurium had virulence effectors that are known to be translocated by Salmonella pathogenicity island 1 (SPI-1)-encoded type III secretion system (T3SS1) into the host cell. In the present study, immunoblot analysis confirmed the secretion of the six T3SS1 effector proteins, namely SipB and SipC (translocators of T3SS1), and SipA, SopA, SopB, and SopE2 (effectors translocated by T3SS1), by OMVs. Results of proteinase K treatment revealed the localization of these T3SS1 effector proteins on the outer surface of OMVs. SipC and SopE2 were secreted by OMVs independent of the three secretion systems T3SS1, T3SS2, and flagella, signifying OMVs to be an alternative delivery system to T3SSs. T3SS1 effectors SipA, SipC, and SopE2 were internalized into the cytoplasm of the host cell by OMVs independent of cellular Salmonella-host cell contact. In epithelial cells, addition of OMVs harboring T3SS1 effectors stimulated the production of F-actin, thereby complementing the attenuated invasion of ΔsopE2 into host cells. These results suggest that S. Typhimurium might exploit OMVs as a long-distance vehicle to deliver T3SS1 effectors into the cytoplasm of the host cell independent of bacteria-host cell interaction.

Salmonella Typhimurium Sensing Strategy Based on the Loop-Mediated Isothermal Amplification Using Retroreflective Janus Particle as a Nonspectroscopic Signaling Probe.

Loop-mediated isothermal amplification (LAMP) is a powerful gene amplification method, which has many advantages, including high specificity, sensitivity, and simple operation. However, quantitative analysis of the amplified target gene with the LAMP assay is very difficult. To overcome this limitation, we developed a novel biosensing platform for molecular diagnosis by integrating the LAMP method and retroreflective Janus particle (RJP) together. The final amplified products of the LAMP assay are dumbbell-shaped DNA structures, containing a single-stranded loop with two different sequences. Therefore, the concentration of the amplified products can be measured in a manner similar to the sandwich-type immunoassay. To carry out the sandwich-type molecular diagnostics using the LAMP product, two DNA probes, with complementary sequences to the loop-regions, were prepared and immobilized on both the sensing surface and the surface of the RJPs. When the amplified LAMP product was applied to the sensing surface, the surface-immobilized DNA probe hybridized to the loop-region of the LAMP product to form a double-stranded structure. When the DNA probe-conjugated RJPs were injected, the RJPs bound to the unreacted loop-region of the LAMP product. The number of RJPs bound to the loop-region of the LAMP product was proportional to the concentration of the amplified LAMP product, indicating that the concentration of the target gene can be quantitatively analyzed by counting the number of observed RJPs. Using the developed system, a highly sensitive and selective quantification of Salmonella was successfully performed with a detection limit of 102 CFU.

Genomic Approaches for Understanding the Characteristics of Salmonella enterica subsp. enterica Serovar Typhimurium ST1120, Isolated from Swine Feces in Korea.

Salmonella enterica subsp. enterica serovar Typhimurium, one of the most common foodborne pathogens, is transmitted mainly through contaminated food derived from infected animals. In this study, S. Typhimurium ST1120, an isolate from pig feces in Korea, was subjected to whole-genome analysis to understand its genomic features associated with virulence. The genome of ST1120 was found to have a circular chromosome of 4,855,001 bp (GC content 52.2%) and a plasmid of 6,863 bp (GC content 46.0%). This chromosome was predicted to have 4,558 open reading frames (ORFs), 17 pseudogenes, 22 rRNA genes, and 86 tRNA genes. Its plasmid was predicted to have three ORFs. Comparative genome analysis revealed that ST1120 was phylogenetically close to S. Typhimurium U288, a critical isolate in piggery farms and food chains in Europe. In silico functional analysis predicted that the ST1120 genome harbored multiple genes associated with virulence and stress resistance, including Salmonella pathogenicity islands (SPIs containing SPI-1 to SPI-5, SPI-13, and SPI-14), C63PI locus, ST104 prophage locus, and various antibiotic resistance genes. In accordance with these analysis results, ST1120 showed competence in invasion and survival abilities when it was added to host cells. It also exhibited robust resistance against antibiotics in comparison with other S. Typhimurium strains. This is the first report of the complete genome sequence of S. Typhimurium isolated from swine in Korea. Comparative genome analysis between ST1120 and other Salmonella strains would provide fruitful information toward understanding Salmonella host specificity and developing control measures against S. Typhimurium infection.

The complete genome sequence of Cronobacter sakazakii ATCC 29544T, a food-borne pathogen, isolated from a child's throat.

BACKGROUND: Cronobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of severe diseases: meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. However, the pathogenesis mechanism of this pathogen remains largely unknown. To determine its pathogenesis at the genomic level, the genome of C. sakazakii ATCC 29544T was completely sequenced and analyzed. RESULTS: The genomic DNA, containing a circular chromosome and three plasmids, is composed of 4,511,265 bp with a GC content of 56.71%, containing 4380 predicted open reading frames (ORFs), 22 rRNA genes, and 83 tRNA genes. The plasmids, designated pCSK29544_p1, pCSK29544_p2, and pCSK29544_p3, were 93,905-bp, 4938-bp, and 53,457-bp with GC contents of 57.02, 54.88, and 50.07%, respectively. They were also predicted to have 72, 6, and 57 ORFs without RNA genes. CONCLUSIONS: The strain ATCC 29544T genome has ompA and ibeB-homologous cusC genes, probably associated with the invasion of human brain microvascular endothelial cells (BMECs). In addition, gene clusters for siderophore production (iucABCD/iutA) and the related transport system (eitCBAD) were detected in pCSK29544_p1 plasmid, indicating better iron uptake ability for survival. Furthermore, to survive under extremely dry condition like milk powder, this genome has gene clusters for biosynthesis of capsular proteins (CSK29544_00281-00284) and cellulose (CSK29544_01124-01127) for biofilm formation and a gene cluster for utilization of sialic acid in the milk (nanKTAR). The genome information of C. sakazakii ATCC 29544T would provide further understanding of its pathogenesis at the molecular level for the regulation of pathogenicity and the development of a rapid detection method using biomarkers.

[Factors affecting national health insurance mass screening participation in the disabled].

OBJECTIVES: As the disabled have higher prevalence rates and earlier onsets of chronic diseases than the non-disabled, their participation in mass screening is important for the early detection and intervention of chronic diseases. Nevertheless, in Korea, the disabled have lower participation rates in mass screening services than the non-disabled. The purpose of the study was to find determinants for the participation in the National Health Insurance (NHI) mass screening program among the disabled. METHODS: In this study, the NHI mass screening data of 423,076 disabled people, which were identified using the National Disability Registry (2003), were analyzed. Of the factors affecting the participation rates in mass screenings, the following variables were included for the analysis: socioeconomic stati, such as sex, age, category of health insurance program, region and income; disability characteristics, such as disability type, and severity. A multiple logistic regression analysis was used to evaluate the association between the participation rates, disability characteristics variables and demographic variables. RESULTS: The participation rate in mass screening of the disabled was 41.3%, but was lower in females, an age of more than 70 years, self-employed and for those with an average monthly insurance premium over 133,500 Won and in metropolitan regions. The participation rate was 1.31 times lower in females than males (95% CI=1.29-1.33); 3.50 times lower in the elderly (more than 70 years) than the younger (95% CI=3.33-3.67); 1.43 times lower in those who live in metropolitan areas (95% CI=1.40-1.46); 2.59 times lower for those in a health insurance program for the self-employed than for employees (95% CI=2.56-2.63); 1.19 times lower for the higher income (more than 133,500) than the lower income group (4,400-22,000) for the average monthly insurance premium (95% CI=1.15-1.23); 2.04 times lower for those with brain palsy and stroke disabilities than with auditory impairments (95% CI= 1.97-2.11) and 3.27 times for those with severe compared to mild disabilities (95% CI=3.15-3.40). CONCLUSIONS: The disabled with high severity, and locomotive and communication disabilities have lower participation rates in mass screening services in Korea.