Constitutive Dicer1 phosphorylation accelerates metabolism and aging in vivo.

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.

IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort.

BACKGROUND: Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. OBJECTIVES: To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. METHODS: This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of ß-lactamase genes, respectively. RESULTS: Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified ß-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing ß-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated ß-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. CONCLUSIONS: These data demonstrate IS26-mediated mechanisms underlying ß-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

Clonal Emergence of Invasive Multidrug-Resistant Staphylococcus epidermidis Deconvoluted via a Combination of Whole-Genome Sequencing and Microbiome Analyses.

Background: Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid. Methods: Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction. Results: Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use. Conclusions: The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.

Identification of a novel CG307 sub-clade in third-generation-cephalosporin-resistant Klebsiella pneumoniae causing invasive infections in the USA.

Despite the notable clinical impact, recent molecular epidemiology regarding third-generation-cephalosporin-resistant (3GC-R) Klebsiella pneumoniae in the USA remains limited. We performed whole-genome sequencing of 3GC-R K. pneumoniae bacteraemia isolates collected from March 2016 to May 2022 at a tertiary care cancer centre in Houston, TX, USA, using Illumina and Oxford Nanopore Technologies platforms. A comprehensive comparative genomic analysis was performed to dissect population structure, transmission dynamics and pan-genomic signatures of our 3GC-R K. pneumoniae population. Of the 178 3GC-R K. pneumoniae bacteraemias that occurred during our study time frame, we were able to analyse 153 (86 %) bacteraemia isolates, 126 initial and 27 recurrent isolates. While isolates belonging to the widely prevalent clonal group (CG) 258 were rarely observed, the predominant CG, 307, accounted for 37 (29 %) index isolates and displayed a significant correlation (Pearson correlation test P value=0.03) with the annual frequency of 3GC-R K. pneumoniae bacteraemia. Interestingly, only 11 % (4/37) of CG307 isolates belonged to the commonly detected 'Texas-specific' clade that has been observed in previous Texas-based K. pneumoniae antimicrobial-resistance surveillance studies. We identified nearly half of our CG307 isolates (n=18) belonged to a novel, monophyletic CG307 sub-clade characterized by the chromosomally encoded bla SHV-205 and unique accessory genome content. This CG307 sub-clade was detected in various regions of the USA, with genome sequences from 24 additional strains becoming recently available in the National Center for Biotechnology Information (NCBI) SRA database. Collectively, this study underscores the emergence and dissemination of a distinct CG307 sub-clade that is a prevalent cause of 3GC-R K. pneumoniae bacteraemia among cancer patients seen in Houston, TX, and has recently been isolated throughout the USA.

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure.

Background: It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. Methods: Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum ß-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/ H 30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. Results: All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P -value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing bla CTX-M-15 copy numbers via modular, insertion sequence 26 (IS 26 ) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. Conclusions: ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/ H 30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

Rapid Whole Genome Characterization of High-Risk Pathogens Using Long-Read Sequencing to Identify Potential Healthcare Transmission.

Objective: Routine use of whole genome sequencing (WGS) has been shown to help identify transmission of pathogens causing healthcare-associated infections (HAIs). However, the current gold standard of short-read, Illumina-based WGS is labor and time-intensive. In light of recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing, we sought to establish a low resource utilization approach capable of providing accurate WGS-based comparisons of HAI pathogens within a time frame allowing for infection prevention and control (IPC) interventions. Methods: WGS was prospectively performed on antimicrobial-resistant pathogens at increased risk of potential healthcare transmission using the ONT MinION sequencer with R10.4.1 flow cells and Dorado basecalling algorithm. Potential transmission was assessed via Ridom SeqSphere+ for core genome multilocus sequence typing and MINTyper for reference-based core genome single nucleotide polymorphisms using previously published cut-off values. The accuracy of our ONT pipeline was determined relative to Illumina-based WGS data generated from the same genomic DNA sample. Results: Over a six-month period, 242 bacterial isolates from 216 patients were sequenced by a single operator. Compared to the Illumina gold-standard data, our ONT pipeline achieved a Q score of 60 for assembled genomes, even with a coverage rate of as low as 40X. The mean time from initiating DNA extraction to complete genetic analysis was 2 days (IQR 2-3.25 days). We identified five potential transmission clusters comprising 21 isolates (8.7% of all sequenced strains). Combining ONT WGS data with epidemiological data, >70% (15/21) of the isolates originated from patients with potential healthcare transmission links. Conclusions: Via a stand-alone ONT pipeline, we detected potentially transmitted HAI pathogens rapidly and accurately, aligning closely with epidemiological data. Our low-resource method has the potential to assist in the efficient detection and deployment of preventative measures against HAI transmission.

Recurrent Somatic Copy Number Alterations and Their Association with Oncogene Expression Levels in High-Grade Ovarian Serous Carcinoma.

Somatic copy number alterations (SCNAs) are frequently observed in high-grade ovarian serous carcinoma (HGOSC). However, their impact on gene expression levels has not been systematically assessed. In this study, we explored the relationship between recurrent SCNA and gene expression using The Cancer Genome Atlas Pan Cancer dataset (OSC, TCGA, PanCancer Atlas) to identify cancer-related genes in HGOSC. We then investigated any association between highly correlated cancer genes and clinicopathological parameters, including age of diagnosis, disease stage, overall survival (OS), and progression-free survival (PFS). A total of 772 genes with recurrent SCNAs were observed. SCNA and mRNA expression levels were highly correlated for 274 genes; 24 genes were classified as a Tier 1 gene in the Cancer Gene Census in the Catalogue of Somatic Mutations in Cancer (CGC-COSMIC). Of these, 11 Tier 1 genes had highly correlated SCNA and mRNA expression levels: TBL1XR1, PIK3CA, UBR5, EIF3E, RAD21, EXT1, RECQL4, KRAS, PRKACA, BRD4, and TPM4. There was no association between gene amplification and disease stage or PFS. EIF3E, RAD21, and EXT1 were more frequently amplified in younger patients, specifically those under the age of 55 years. Patients with tumors carrying PRKACA, BRD4, or TPM4 amplification were associated with a significantly shorter OS. RECQL4 amplification was more frequent in younger patients, and tumors with this amplification were associated with a significantly better OS.

Temporal dynamics of genetically heterogeneous extended-spectrum cephalosporin-resistant Escherichia coli bloodstream infections.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESC-R-Ec) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R-Ec infections. To address the paucity of recent ESC-R-Ec molecular epidemiology data in the United States, we used whole-genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R-Ec at a tertiary care cancer center in Houston, Texas, collected from 2016 to 2020. During the study time frame, there were 1,154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R-Ec. Using time series analyses, we identified a temporal dynamic of ESC-R-Ec distinct from ESC-susceptible E. coli (ESC-S-Ec), with cases peaking in the last 6 months of the calendar year. WGS of 297 ESC-R-Ec strains revealed that while STc131 strains accounted for ~45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R-Ec clonal complexes. bla CTX-M variants accounted for most ß-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R-Ec), and amplification of bla CTX-M genes was widely detected in ESC-R-Ec strains, particularly in carbapenem non-susceptible, recurrent BSI strains. Bla CTX-M-55 was significantly enriched within phylogroup A strains, and we identified bla CTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R-Ec infections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens. IMPORTANCE Given that E. coli is the leading cause of worldwide ESC-R Enterobacterales infections, we sought to assess the current molecular epidemiology of ESC-R-Ec using a WGS analysis of many BSIs over a 5-year period. We identified fluctuating temporal dynamics of ESC-R-Ec infections, which have also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited but genetically diverse group of ESC-R-Ec clonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of ß-lactamase gene copy number in ESC-R-Ec infections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R-Ec strains. These data suggest that serious ESC-R-Ec infections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

Temporal Dynamics of Genetically Heterogeneous Extended-Spectrum Cephalosporin Resistant Escherichia coli Bloodstream Infections.

Extended-spectrum cephalosporin resistant Escherichia coli (ESC-R- Ec ) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R- Ec infections. To address the paucity of recent ESC-R- Ec molecular epidemiology data in the United States, we used whole genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R- Ec at a tertiary care cancer center in Houston, Texas collected from 2016-2020. During the study timeframe, there were 1154 index E. coli bloodstream infections (BSIs) of which 389 (33.7%) were ESC-R- Ec . Using time series analyses, we identified a temporal dynamic of ESC-R- Ec distinct from ESC-susceptible E. coli (ESC-S- Ec ), with cases peaking in the last six months of the calendar year. WGS of 297 ESC-R- Ec strains revealed that while STc131 strains accounted for ∼45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R- Ec clonal complexes. Bla CTX-M variants accounted for most ß-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R -Ec ), and amplification of bla CTX-M genes was widely detected in ESC-R- Ec strains, particularly in carbapenem non-susceptible, recurrent BSI strains. Bla CTX-M-55 was significantly enriched within phylogroup A strains, and we identified bla CTX-M-55 plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R- Ec infections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens. IMPORTANCE: Given that E. coli is the leading cause of worldwide ESC-R Enterobacterales infections, we sought to assess the current molecular epidemiology of ESC-R- Ec using a WGS analysis of many BSIs over a five-year period. We identified fluctuating temporal dynamics of ESC-R- Ec infections, which has also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited, but genetically diverse group of ESC-R- Ec clonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of ß-lactamase gene copy number in ESC-R- Ec infections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R- Ec strains. These data suggest that serious ESC-R- Ec infections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa.

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.

A Dot/Icm-translocated ankyrin protein of Legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa.

The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth.

Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages.

Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.

Helicobacter pylori evolution: lineage- specific adaptations in homologs of eukaryotic Sel1-like genes.

Geographic partitioning is postulated to foster divergence of Helicobacter pylori populations as an adaptive response to local differences in predominant host physiology. H. pylori's ability to establish persistent infection despite host inflammatory responses likely involves active management of host defenses using bacterial proteins that may themselves be targets for adaptive evolution. Sequenced H. pylori genomes encode a family of eight or nine secreted proteins containing repeat motifs that are characteristic of the eukaryotic Sel1 regulatory protein, whereas the related Campylobacter and Wolinella genomes each contain only one or two such "Sel1-like repeat" (SLR) genes ("slr genes"). Signatures of positive selection (ratio of nonsynonymous to synonymous mutations, dN/dS = omega > 1) were evident in the evolutionary history of H. pylori slr gene family expansion. Sequence analysis of six of these slr genes (hp0160, hp0211, hp0235, hp0519, hp0628, and hp1117) from representative East Asian, European, and African H. pylori strains revealed that all but hp0628 had undergone positive selection, with different amino acids often selected in different regions. Most striking was a divergence of Japanese and Korean alleles of hp0519, with Japanese alleles having undergone particularly strong positive selection (omegaJ > 25), whereas alleles of other genes from these populations were intermingled. Homology-based structural modeling localized most residues under positive selection to SLR protein surfaces. Rapid evolution of certain slr genes in specific H. pylori lineages suggests a model of adaptive change driven by selection for fine-tuning of host responses, and facilitated by geographic isolation. Characterization of such local adaptations should help elucidate how H. pylori manages persistent infection, and potentially lead to interventions tailored to diverse human populations.

Identification of a chimeric emm gene and novel emm pattern in currently circulating strains of emm4 Group A Streptococcus.

Group A Streptococcus (GAS) is classified on the basis of the sequence of the gene encoding the M protein (emm) and the patterns into which emm types are grouped. We discovered a novel emm pattern in emm4 GAS, historically considered pattern E, arising from a fusion event between emm and the adjacent enn gene. We identified the emm-enn fusion event in 51 out of 52 emm4 GAS strains isolated by national surveillance in 2015. GAS isolates with an emm-enn fusion event completely replaced pattern E emm4 strains over a 4-year span in Houston (2013-2017). The novel emm-enn gene fusion and new emm pattern has potential vaccine implications.

Hypervirulent group A Streptococcus emergence in an acaspular background is associated with marked remodeling of the bacterial cell surface.

Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated-a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.

Identification and Whole Genome Sequencing of the First Case of Kosakonia radicincitans Causing a Human Bloodstream Infection.

The taxonomy of Enterobacter species is rapidly changing. Herein we report a bloodstream infection isolate originally identified as Enterobacter cloacae by Vitek2 methodology that we found to be Kosakonia radicincitans using genetic means. Comparative whole genome sequencing of our isolate and other published Kosakonia genomes revealed these organisms lack the AmpC ß-lactamase present on the chromosome of Enterobacter sp. A fimbriae operon primarily found in Escherichia coli O157:H7 isolates was present in our organism and other available K. radicincitans genomes. This is the first report of a Kosakonia species, which are typically associated with plants, causing a human infection.

Divergent evolution of Di-lysine ER retention vs. farnesylation motif-mediated anchoring of the AnkB virulence effector to the Legionella-containing vacuolar membrane.

Within macrophages and amoeba, the Legionella-containing vacuole (LCV) membrane is derived from the ER. The bona fide F-box AnkB effector protein of L. pneumophila strain AA100/130b is anchored to the cytosolic side of the LCV membrane through host-mediated farnesylation of its C-terminal eukaryotic "CaaX" motif. Here we show that the AnkB homologue of the Paris strain has a frame shift mutation that led to a loss of the CaaX motif and a concurrent generation of a unique C-terminal KNKYAP motif, which resembles the eukaryotic di-lysine ER-retention motif (KxKxx). Our phylogenetic analyses indicate that environmental isolates of L. pneumophila have a potential positive selection for the ER-retention KNKYAP motif. The AnkB-Paris effector is localized to the LCV membrane most likely through the ER-retention motif. Its ectopic expression in HEK293T cells localizes it to the perinuclear ER region and it trans-rescues the ankB mutant of strain AA100/130b in intra-vacuolar replication. The di-lysine ER retention motif of AnkB-Paris is indispensable for function; most likely as an ER retention motif that enables anchoring to the ER-derived LCV membrane. Our findings show divergent evolution of the ankB allele in exploiting either host farnesylation or the ER retention motif to be anchored into the LCV membrane.

Helicobacter pylori adhesion to carbohydrates.

Adherence of bacterial pathogens to host tissues contributes to colonization and virulence and typically involves specific interactions between bacterial proteins called adhesins and cognate oligosaccharide (glycan) or protein motifs in the host that are used as receptors. A given pathogen may have multiple adhesins, each specific for a different set of receptors and, potentially, with different roles in infection and disease. This chapter provides strategies for identifying and analyzing host glycan receptors and the bacterial adhesins that exploit them as receptors, with particular reference to adherence of the gastric pathogen Helicobacter pylori.

Complete Genome Sequence of Streptococcus mitis Strain SVGS_061 Isolated from a Neutropenic Patient with Viridans Group Streptococcal Shock Syndrome.

Streptococcus mitisfrequently causes invasive infections in neutropenic cancer patients, with a subset of patients developing viridans group streptococcal (VGS) shock syndrome. We report here the first complete genome sequence ofS. mitisstrain SVGS_061, which caused VGS shock syndrome, to help elucidate the pathogenesis of severe VGS infection.

Draft Genome Sequences of Four Genetically Distinct Human Isolates of Streptococcus dysgalactiae subsp. equisimilis.

ß-Hemolytic group C and group G streptococci (GCS-GGS; Streptococcus dysgalactiae subsp. equisimilis) emerged as human pathogens in the late 1970s. We report here the draft genome sequences of four genetically distinct human strains of GCS-GGS isolated between the 1960s and 1980s. Comparative analysis of these genomes may provide a deeper understanding of GCS-GGS genome and virulence evolution.