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Abstract Superantigens (sAgs) are bacterial virulence factors that induce a state of immune hyperactivation by forming a bridge between certain subsets of T cell receptor (TCR) β chains on T lymphocytes, and class II major histocompatibility complex (MHC-II) molecules; this cross-linking leads to indiscriminate T cell activation, cytokine storm and toxic shock. Here we show that sAg exposure drives the preferential expansion of naive and central memory T cell subsets, but not effector or resident memory T cells, which instead, hyper release pro-inflammatory cytokines. A targeted therapeutic approach to minimise cytokine release by effector memory T cells attenuated sAg-induced cytokine release. Irrespective of antigen experience, sAg activation does not render mature T cells permanently dysfunctional, and full restoration of effector function is observed following a transient and reversible anergy. Moreover, we show that in the face of sAg induced immune hyperactivation, an intact bacterium-specific CD4+ T cell response can be mounted.

Abstract Gephyrin is thought to play a critical role in clustering glycine receptors at synapses within the central nervous system (CNS). The main in vivo evidence for this comes from Gephyrin (Gphn)-null mice, where glycine receptors are depleted from synaptic regions. However, these mice die at birth, possibly due to impaired molybdenum cofactor (MoCo) synthesis, an essential role Gephyrin assumes throughout an animal. This complicates the interpretation of synaptic phenotypes in Gphn-null mice and raises the question whether the synaptic and enzymatic functions of Gephyrin can be investigated separately. Here, we generated a gephyrinb zebrafish mutant, vo84, that almost entirely lacks Gephyrin staining in the spinal cord. gephyrinb vo84 mutants exhibit normal gross morphology at both larval and adult stages. In contrast to Gphn-null mice, gephyrinb vo84 mutants exhibit normal motor activity and MoCo-dependent enzyme activity. Instead, gephyrinb vo84 mutants display impaired rheotaxis and increased mortality in late development. To investigate what may mediate these defects in gephyrinb vo84 mutants, we examined the cell density of neurons and myelin in the spinal cord and found no obvious changes. Surprisingly, in gephyrinb vo84 mutants, glycine receptors are still present in the synaptic regions. However, their abundance is reduced, potentially contributing to the observed defects. These findings challenge the notion that Gephyrin is absolutely required to cluster glycine receptors at synapses and reveals a new role of Gephyrin in regulating glycine receptor abundance and rheotaxis. They also establish a powerful new model for studying the mechanisms underlying synaptic, rather than enzymatic, functions of Gephyrin.

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

Abstract The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6’)-aph(2”)) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.

Staphylococcus aureus bacteremia causes significant morbidity and mortality. Treatment of staphylococcal infections is hindered by widespread antibiotic resistance, and attempts to develop an S. aureus vaccine have failed. Improved S. aureus treatment and infection prevention options require a deeper understanding of the correlates of protective immunity. CD4+ T cells have been identified as key orchestrators in the defense against S. aureus, but uncertainties persist regarding the subset, polarity, and breadth of the memory CD4+ T-cell pool required for protection. Here, using a mouse model of systemic S. aureus infection, we discovered that the breadth of bacterium-specific memory CD4+ T-cell pool is a critical factor for protective immunity against invasive S. aureus infections. Seeding mice with a monoclonal bacterium-specific circulating memory CD4+ T-cell population failed to protect against systemic S. aureus infection; however, the introduction of a polyclonal and polyfunctional memory CD4+ T-cell pool significantly reduced the bacterial burden. Our findings support the development of a multi-epitope T-cell-based S. aureus vaccine, as a strategy to mitigate the severity of S. aureus bacteremia.

ABSTRACTAmong the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.IMPORTANCEThe opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

The extracellular regions (ECRs) of adhesion GPCRs have diverse biological functions, but their structures and mechanisms of action remain unclear. Here, the authors solve the ECR structure of the Gpr126 receptor and show that ECR conformation and signaling functions are regulated by alternative splicing.

WalKR is an essential two-component regulator that controls peptidoglycan synthesis in the human pathogen Staphylococcus aureus. Here, the authors provide biochemical, structural, and functional evidence supporting that the binding of a zinc ion inhibits autophosphorylation and thus alters WalKR regulatory activity.

The authors find that deletion from Schwann cells of an E3 ubiquitin ligase component called Fbxw7 leads to a phenotype reminiscent of myelination in the central nervous system where a single oligodendrocyte ensheaths multiple axons.

ABSTRACT Antistaphylococcal penicillins such as oxacillin are the key antibiotics in the treatment of invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections; however, mec gene-independent resistance adaptation can cause treatment failure. Despite its clinical relevance, the basis of this phenomenon remains poorly understood. Here, we investigated the genomic adaptation to oxacillin at an unprecedented scale using a large collection of 503 clinical mec-negative isolates and 30 in vitro-adapted isolates from independent oxacillin exposures. By combining comparative genomics, evolutionary convergence, and genome-wide association analysis, we found 21 genetic loci associated with low-level oxacillin resistance, underscoring the polygenic nature of this phenotype. Evidence of adaptation was particularly strong for the c-di-AMP signal transduction pathways (gdpP and dacA) and in the clpXP chaperone-protease complex. The role of mutations in gdpP in conferring low-level oxacillin resistance was confirmed by allele-swapping experiments. We found that resistance to oxacillin emerges at high frequency in vitro (median, 2.9 × 10−6; interquartile range [IQR], 1.9 × 10−6 to 3.9 × 10−6), which is consistent with a recurrent minimum inhibitory concentration (MIC) increase across the global phylogeny of clinical isolates. Nevertheless, adaptation in clinical isolates appears sporadically, with no stably adapted lineages, suggesting a high fitness cost of resistance, confirmed by growth assessment of mutants in rich media. Our data provide a broader understanding of the emergence and dynamics of oxacillin resistance adaptation in S. aureus and a framework for future surveillance of this clinically important phenomenon. IMPORTANCE The majority of Staphylococcus aureus strains causing human disease are methicillin-susceptible (MSSA) and can be treated with antistaphylococcal penicillins (such as oxacillin). While acquisition of the mec gene represents the main resistance mechanism to oxacillin, S. aureus can acquire low-level resistance through adaptive mutations in other genes. In this study, we used genomic approaches to understand the basis of S. aureus adaption to oxacillin and its dynamic at the population level. By combining a genome analysis of clinical isolates from persistent MSSA infections, in vitro selection of oxacillin resistance, and genome-wide association analysis on a large collection of isolates, we identified 21 genes linked to secondary oxacillin resistance. Adaptive mutations in these genes were easy to select when S. aureus was exposed to oxacillin, but they also came at a substantial cost in terms of bacterial fitness, suggesting that this phenotype emerges preferentially in the setting of sustained antibiotic exposure.

ABSTRACT Staphylococcus aureus contains a repertoire of at least 50 and possibly 500 small RNAs (sRNAs). The functions of most sRNAs are not understood, although some are known to respond to environmental changes, including the presence of antibiotics. Here, in an effort to better understand the roles of sRNAs in the context of antibiotic exposure, we took a clinical methicillin-resistant S. aureus (MRSA) isolate and separately deleted eight sRNAs that were significantly upregulated in response to the last-line antibiotic linezolid as revealed by transcriptome sequencing (RNA-seq) comparisons. We also deleted an additional 10 sRNAs that were either highly expressed or previously found to respond to antibiotic exposure. There were no significant changes for any of the 18 mutants in a variety of phenotypic screens, including MIC screens, growth competition assays in the presence of linezolid, biofilm formation, and resistance to whole-blood killing. These data suggest sRNA functional redundancy, because despite their high expression levels upon antibiotic exposure, individual sRNA genes do not affect readily observable bacterial phenotypes. The sRNA transcriptional changes we measured during antibiotic exposure might also reflect sRNA “indifference,” that is, a general stress response not specifically related to sRNA function. These data underscore the need for sensitive assays and new approaches to try and decipher the functions of sRNA genes in S. aureus. IMPORTANCE Bacterial small RNAs (sRNAs) are RNA molecules that can have important regulatory roles across gene expression networks. There is a growing understanding of the scope and potential breadth of impact of sRNAs on global gene expression patterns in Staphylococcus aureus, a major human pathogen. Here, transcriptome comparisons were used to examine the roles of sRNA genes with a potential role in the response of S. aureus to antibiotic exposure. Although no measurable impact on key bacterial phenotypes was observed after deleting each of 18 sRNAs identified by these comparisons, this research is significant because it underscores the subtle modes of action of these sometimes abundant molecules within the bacterium.

ABSTRACT Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus. Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis. Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidis hsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies. IMPORTANCE Staphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus. Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.

ABSTRACT Teixobactin is a new antimicrobial of significant interest. It is active against a number of multidrug-resistant pathogens, including Staphylococcus aureus and Enterococcus faecalis, with no reported mechanisms of teixobactin resistance. However, historically, mechanisms of resistance always exist and arise upon introduction of a new antimicrobial into a clinical setting. Therefore, for teixobactin to remain effective long term, we need to understand how mechanisms of resistance could develop. Here we demonstrate that E. faecalis shows a remarkable intrinsic tolerance to high concentrations of teixobactin. This is of critical importance, as antimicrobial tolerance has been shown to precede the development of antimicrobial resistance. To identify potential pathways responsible for this tolerance, we determined the genomewide expression profile of E. faecalis strain JH2-2 in response to teixobactin using RNA sequencing. A total of 573 genes were differentially expressed (2.0-fold log2 change in expression) in response to teixobactin, with genes involved in cell wall biogenesis and division and transport/binding being among those that were the most upregulated. Comparative analyses of E. faecalis cell wall-targeting antimicrobial transcriptomes identified CroRS, LiaRS, and YclRK to be important two-component regulators of antimicrobial-mediated stress. Further investigation of CroRS demonstrated that deletion of croRS abolished tolerance to teixobactin and to other cell wall-targeting antimicrobials. This highlights the crucial role of CroRS in controlling the molecular response to teixobactin. IMPORTANCE Teixobactin is a new antimicrobial with no known mechanisms of resistance. Understanding how resistance could develop will be crucial to the success and longevity of teixobactin as a new potent antimicrobial. Antimicrobial tolerance has been shown to facilitate the development of resistance, and we show that E. faecalis is intrinsically tolerant to teixobactin at high concentrations. We subsequently chose E. faecalis as a model to elucidate the molecular mechanism underpinning teixobactin tolerance and how this may contribute to the development of teixobactin resistance.

Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of α-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of α-toxin, and triggered limited tissue damage. α-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure α-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of α-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of α-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against α-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of α-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.

Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied class of the GPCR superfamily. aGPCRs are involved in many developmental processes and immune and synaptic functions, but the mode of their signal transduction is unclear. Here, we show that a short peptide sequence (termed the Stachel sequence) within the ectodomain of two aGPCRs (GPR126 and GPR133) functions as a tethered agonist. Upon structural changes within the receptor ectodomain, this intramolecular agonist is exposed to the seven-transmembrane helix domain, which triggers G protein activation. Our studies show high specificity of a given Stachel sequence for its receptor. Finally, the function of Gpr126 is abrogated in zebrafish with a mutated Stachel sequence, and signaling is restored in hypomorphic gpr126 zebrafish mutants upon exogenous Stachel peptide application. These findings illuminate a mode of aGPCR activation and may prompt the development of specific ligands for this currently untargeted GPCR family.

Microglia are phagocytic cells that form the basis of the brain’s immune system. They derive from primitive macrophages that migrate into the brain during embryogenesis, but the genetic control of microglial development remains elusive. Starting with a genetic screen in zebrafish, we show that the noncanonical NOD-like receptor (NLR) nlrc3-like is essential for microglial formation. Although most NLRs trigger inflammatory signaling, nlrc3-like acts cell autonomously in microglia precursor cells to suppress unwarranted inflammation in the absence of overt immune challenge. In nlrc3-like mutants, primitive macrophages initiate a systemic inflammatory response with increased proinflammatory cytokines and actively aggregate instead of migrating into the brain to form microglia. NLRC3-like requires both its pyrin and NACHT domains, and it can bind the inflammasome component apoptosis-associated speck-like protein. Our studies suggest that NLRC3-like may regulate the inflammasome and other inflammatory pathways. Together, these results demonstrate that NLRC3-like prevents inappropriate macrophage activation, thereby allowing normal microglial development.

Background The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an “African” CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide. Methods We first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages. Results We show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCCmec-IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isolates compared with the other S. aureus lineages. The Ghanaian ST88 isolates had only 658 core genome SNPs and there was no correlation between phylogeny and geography, suggesting the recent spread of this clone. The lineage was also resistant to multiple classes of antibiotics including β-lactams, tetracycline and chloramphenicol. Discussion This study reveals that S. aureus ST88-IV is a recently emerging and rapidly spreading CA-MRSA clone in Ghana. The study highlights the capacity of small snapshot genomic studies to provide actionable public health information in resource limited settings. To our knowledge this is the first genomic assessment of the ST88 CA-MRSA clone.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

SUMMARY Mutations in SEC63 cause polycystic liver disease in humans. Sec63 is a member of the endoplasmic reticulum (ER) translocon machinery, although it is unclear how mutations in SEC63 lead to liver cyst formation in humans. Here, we report the identification and characterization of a zebrafish sec63 mutant, which was discovered in a screen for mutations that affect the development of myelinated axons. Accordingly, we show that disruption of sec63 in zebrafish leads to abnormalities in myelinating glia in both the central and peripheral nervous systems. In the vertebrate nervous system, segments of myelin are separated by the nodes of Ranvier, which are unmyelinated regions of axonal membrane containing a high density of voltage-gated sodium channels. We show that sec63 mutants have morphologically abnormal and reduced numbers of clusters of voltage-gated sodium channels in the spinal cord and along peripheral nerves. Additionally, we observed reduced myelination in both the central and peripheral nervous systems, as well as swollen ER in myelinating glia. Markers of ER stress are upregulated in sec63 mutants. Finally, we show that sec63 mutants develop liver pathology. As in glia, the primary defect, detectable at 5 dpf, is fragmentation and swelling of the ER, indicative of accumulation of proteins in the lumen. At 8 dpf, ER swelling is severe; other pathological features include disrupted bile canaliculi, altered cytoplasmic matrix and accumulation of large lysosomes. Together, our analyses of sec63 mutant zebrafish highlight the possible role of ER stress in polycystic liver disease and suggest that these mutants will serve as a model for understanding the pathophysiology of this disease and other abnormalities involving ER stress.

ABSTRACT Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages. IMPORTANCE The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus.