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.Peer Reviewe

bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection.

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells

Genome Sequence of Paracoccus contaminans LMG 29738T, Isolated from a Water Microcosm

We announce here the complete genome sequence of Paracoccus contaminans LMG 29738T, which we recently isolated from a contaminated water microcosm. The genome consists of a 2.94-Mb chromosome and a 94-kb plasmid. To our knowledge, we provide the first DNA methylation analysis of a Paracoccus species

Two Novel EHEC/EAEC Hybrid Strains Isolated from Human Infections

The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H−, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H−, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized

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

A patatin‐like phospholipase functions during gametocyte induction in the malaria parasite Plasmodium falciparum

International audienc

Toxicity of the two novel EHEC/EAEC strains towards Vero cells.

<p>As a positive control EHEC EDL933 and as negative controls <i>E. coli</i> K12c600 and EAEC 042 were incubated with Vero cells. Further EHEC/EAEC 11-02027 (O104:H4 outbreak 2011), EHEC/EAEC 10-06235 (O59:H⁻), EHEC/EAEC 12-05829 (Orough:H⁻), and EAEC 11-08343 (O59:H⁻) were analyzed. Toxicity of EHEC EDL933 served as a quantitative reference and was set to 100%. Bars represent means and standard deviations of triplicate samples and 256-fold diluted culture preparations. Asterisks indicate significantly lower cytotoxicity compared to the EDL933 reference strain, however all EHEC/EAEC strains were significantly more cytotoxic then the <i>E. coli</i> K12 control, the EAEC 042, as well as EAEC 11-08343 (O59:H⁻) strains (two tailed student’s t-test type 1, p<0.01; p<0.03 for EHEC/EAEC 12-05829 compared to <i>E. coli</i> K12).</p

Pulsed-field gel analysis after XbaI macrorestriction shows that the EHEC/EAEC O59:H⁻ (upper lane) and EAEC O59:H⁻ (lower lane) are not closely related.

<p>Pulsed-field gel analysis after XbaI macrorestriction shows that the EHEC/EAEC O59:H⁻ (upper lane) and EAEC O59:H⁻ (lower lane) are not closely related.</p

Plasmid profile of the two novel EHEC/EAEC strains and identification of the pAA plasmid.

<p>(<b>A</b>) EHEC/EAEC 12-05829 (Orough:H⁻), EAEC 11-08343 (O59:H⁻), EHEC/EAEC 10-06235 (O59:H⁻), EHEC/EAEC 11-02027 (O104:H4 outbreak 2011), and EHEC EDL933 (O157:H7) were analyzed for their plasmid profile. <i>E. coli</i> reference strain 39R861 plasmids served as molecular mass standard. (<b>B</b>) Southern hybridization with plasmid DNA of the same strains as mentioned in (A) was performed using a digoxigenin-labelled <i>aatA</i> gene probe. *EHEC/EAEC; ?pAA plasmid.</p