Search

Legionella pneumophilagrows 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 Δsdestrains. Mutations in thesdhA,ridLandlegA3genes aggravated the Δsdefitness defect, resulting in disruption of theLegionella-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 statementMaintaining replication compartment integrity is critical for growth of intravacuolar pathogens within host cells. By identifying genetically redundant pathways,Legionella pneumophilaSde 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 theL. pneumophilareplicative niche.

Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for establishing an ecological niche necessary for epidemic outbreaks in humans. 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 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 WT, 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.ImportanceWater 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 Legionnaire’s 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 crosslinking 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.

As the supply of agricultural machinery has increases, the distribution of used machinery also increases. However, the legal system for their distribution is insufficient. The objective of this study was to develop an improved distribution system. The first step in achieving this objective involved establishing a framework based on the analysis of current laws in Korea, alongside the distribution systems for both used agricultural machinery in Japan and used cars in Korea. The second stage included a survey on the distribution of used agricultural machinery from 28 dealers and 43 farmers, with the results informing the details of the framework. In the distribution systems for used agricultural machinery in Japan or used cars in Korea, dealers are registered and publicly announced, and there are dealer management organizations. The survey results showed that 91.3% of the dealers conducted pre-sale maintenance, 81.5% provided a warranty, and 85.7% were willing to apply for registration in the improved distribution system. It is necessary to register and publicly announce dealers of used agricultural machinery and to establish a management organization. Moreover, registered dealers must perform pre-sale maintenance and provide a warranty to secure the quality of the goods. Furthermore, in the mid-to-long term, the management organization facilitates the distribution of used agricultural machinery and strengthens the competitiveness of the agricultural machinery industry by increasing exports, recycling used parts, and publishing price guidebooks.

SUMMARY 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., Graphical Abstract, In brief The Legionella SdhA protein supports intracellular growth in both mammalian macrophages and amoebae by preventing disintegration of the membrane-encompassed replication compartment. Choi et al. show that SdhA binds OculoCerebroRenal syndrome of Lowe (OCRL) protein, preventing early endosomal compartments of the host cell from docking and destabilizing the replication compartment.

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.

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.

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.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13099-016-0150-0) contains supplementary material, which is available to authorized users.

Additional file 1: Figure S1. The morphology of C. sakazakii ATCC 29544 imaged by energy-filtering transmission electron microscopy (EF-TEM). EF-TEM photograph was obtained by 2% uranyl acetate on copper grids and examined with ET-TEM at a voltage of 120 kV (LIBRA 120, Zeiss, Oberkochen, Germany). Figure S2: Comparative analysis of two ATCC 29544-specific gene clusters: (A) lac operon and (B) arsenic resistance. The amino acid sequence identities between associated genes are indicated as percentages.

A boundary sequence is a good representation of arbitrary shaped regions, but not directly used in computing shape features such as area, centroid, orientation, and so forth. In this paper we show that the shape features can be easily computed by using cross-sections derived from a boundary sequence. The cross-sections are vertical line segments in the region and can be determined by tracing the boundary sequence once. Furthermore, a boundary sequence extraction method is also proposed, which generates a boundary sequence for each region in a binary image by scanning the image only once. The proposed method works well even if a region has holes.