A conserved signal-peptidase antagonist modulates membrane homeostasis of actinobacterial sortase critical for surface morphogenesis.

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium Actinomyces oris. Genetic deletion of safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by srtA depletion. The pleiotropic defect of the safA mutant is rescued by ectopic expression of safA from not only A. oris, but also Corynebacterium diphtheriae or Corynebacterium matruchotii. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the safA mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.

Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes.

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.

Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence in Enterococcus faecalis.

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host-pathogen interactions.

Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope.

In Gram-positive bacteria, protein precursors with a signal peptide and a cell wall sorting signal (CWSS)-which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues-are targeted to the cell envelope by a transpeptidase enzyme call sortase. Evolution and selective pressure gave rise to six classes of sortase, i.e., SrtA-F. Only class C sortases are capable of polymerizing substrates harboring the pilin motif and CWSS into protein polymers known as pili or fimbriae, whereas the others perform cell wall anchoring functions. Regardless of the products generated from these sortases, the basic principle of sortase-catalyzed transpeptidation is the same. It begins with the cleavage of the LPXTG motif, followed by the cross-linking of this cleaved product at the threonine residue to a nucleophile, i.e., an active amino group of the peptidoglycan stem peptide or the lysine residue of the pilin motif. This chapter will summarize the efforts to identify and characterize sortases and their associated pathways with emphasis on the cell wall anchoring function.

Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris.

Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial vitamin K epoxide reductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent ß-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class ActinobacteriaIMPORTANCE It has recently been shown in the high-GC Gram-positive bacteria (or Actinobacteria) Actinomyces oris and Corynebacterium diphtheriae that oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. In A. oris, reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that the A. oris membrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and biofilm formation and appears to be conserved in members of the class Actinobacteria, including Mycobacterium tuberculosis.

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris.

UNLABELLED: The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. IMPORTANCE: Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal "glyco-stress" caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases.

The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly.

The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei, an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei.

Evaluation of glycogen storage disease as a cause of ketotic hypoglycemia in children.

INTRODUCTION: Ketone formation is a normal response when hypoglycemia occurs. Since the majority of children with recurrent hypoglycemia cannot be diagnosed with a known endocrine or metabolic disorder on a critical sample, ketotic hypoglycemia has been described as the most common cause of low blood glucose concentrations in children. Critical samples, however, will miss the ketotic forms of glycogen storage disease (GSD), which present with elevated ketones, hypoglycemia, and normal hormonal concentrations. RESULTS: A total of 164 children (96 boys, 68 girls) were enrolled in the study. Prediction of pathogenicity of DNA changes using computer modeling confirmed pathology in 20 individuals [four GSD 0, two GSD VI, 12 GSD IX alpha, one GSD IX beta, one GSD IX gamma] (12%). Boys were most likely to have changes in the PHKA2 gene, consistent with GSD IX alpha, an X-linked disorder. CONCLUSIONS: Mutations in genes involved in glycogen synthesis and degradation were commonly found in children with idiopathic ketotic hypoglycemia. GSD IX is likely an unappreciated cause of ketotic hypoglycemia in children, while GSD 0 and VI are relatively uncommon. GSD IX alpha should particularly be considered in boys with unexplained hypoglycemia.

Applications of designer phage encoding recombinant gene payloads.

Advances in genetic engineering, synthetic biology, and DNA sequencing have transformed the re-emergent therapeutic bacteriophage field. The increasing rate of multidrug resistant (MDR) infections and the speed at which new bacteriophages can be isolated, sequenced, characterized, and engineered has reinvigorated phage therapy and unlocked new applications of phages for modulating bacteria. The methods used to genetically engineer bacteriophages are undergoing significant development, but identification of heterologous gene payloads with desirable activity and determination of their impact on bacteria or human cells in translationally relevant applications remain underexplored areas. Here, we discuss and categorize recombinant gene payloads for their potential outcome on phage-bacteria interactions when genetically engineered into phage genomes for expression in their bacterial hosts.

Aggressive therapy improves cirrhosis in glycogen storage disease type IX.

Glycogen storage disease type IX (GSD IX) is described as a benign condition that often does not require treatment. Most patients with the disease are thought to outgrow the childhood manifestations, which include hepatomegaly, poor growth, and ketosis with or without hypoglycemia. Long term complications including fibrosis and cirrhosis have seldom been reported in the most common subtype, GSD IXα. We present two cases of children with GSD IXα who had fibrosis at the time of diagnosis in addition to the commonly reported disease manifestations. Structured therapy with frequent doses of uncooked cornstarch and protein supplementation was initiated, and both children responded with improved growth velocity, increased energy, decreased hepatomegaly and improved well-being. Additionally, radiographic features of fibrosis improved. We propose that GSD IXα is not a benign condition. Even in patients with a less severe presentation, consideration of a structured treatment regimen to improve quality of life appears warranted.

Membrane-localized expression, production and assembly of Vibrio parahaemolyticus T3SS2 provides evidence for transertion.

It has been proposed that bacterial membrane proteins may be synthesized and inserted into the membrane by a process known as transertion, which involves membrane association of their encoding genes, followed by coupled transcription, translation and membrane insertion. Here, we provide evidence supporting that the pathogen Vibrio parahaemolyticus uses transertion to assemble its type III secretion system (T3SS2), to inject virulence factors into host cells. We propose a two-step transertion process where the membrane-bound co-component receptor (VtrA/VtrC) is first activated by bile acids, leading to membrane association and expression of its target gene, vtrB, located in the T3SS2 pathogenicity island. VtrB, the transmembrane transcriptional activator of T3SS2, then induces the localized expression and membrane assembly of the T3SS2 structural components and its effectors. We hypothesize that the proposed transertion process may be used by other enteric bacteria for efficient assembly of membrane-bound molecular complexes in response to extracellular signals.

A Cell-based Screen in Actinomyces oris to Identify Sortase Inhibitors.

Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.

Structure and Mechanism of LcpA, a Phosphotransferase That Mediates Glycosylation of a Gram-Positive Bacterial Cell Wall-Anchored Protein.

The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-ß-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms.IMPORTANCE In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.

Biogenesis of the Gram-positive bacterial cell envelope.

The Gram-positive cell envelope serves as a molecular platform for surface display of capsular polysaccharides, wall teichoic acids (WTAs), lipoteichoic acids (LTAs), lipoproteins, surface proteins and pili. WTAs, LTAs, and sortase-assembled pili are a few features that make the Gram-positive cell envelope distinct from the Gram-negative counterpart. Interestingly, a set of LytR-CpsA-Psr family proteins, found in all Gram-positives but limited to a minority of Gram-negative organisms, plays divergent functions, while decorating the cell envelope with glycans. Furthermore, a phylum of Gram-positive bacteria, the actinobacteria, appear to employ oxidative protein folding as the major folding mechanism, typically occurring in an oxidizing environment of the Gram-negative periplasm. These distinctive features will be highlighted, along with recent findings in the cell envelope biogenesis.

MglA/SspA complex interactions are modulated by inorganic polyphosphate.

The transcription factors MglA and SspA of Francisella tularensis form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the Francisella pathogenicity island (FPI) genes. These genes are essential for this pathogen's virulence and survival within host cells. Our goal was to determine if an intracellular metabolite modulate these protein/protein interactions. In this study, we identified inorganic polyphosphate (polyP) as a signal molecule that promotes the interaction of MglA and SspA from F. tularensis SCHU S4. Analysis of the Mgla/SspA interaction was carried out using a two-hybrid system. The Escherichia coli reporter strain contained a deletion on the ppK-ppX operon, inhibiting polyP synthesis. The interaction between MglA and SspA was significantly impaired, as was the interaction between the MglA/SspA complex and the regulatory protein, FevR, indicating the stabilizing effect of polyP. In F. tularensis, chromatin immune precipitation studies revealed that in the absence of polyP, binding of the MglA/SspA complex to the promoter region of the pdpD, iglA, fevR and ppK genes is decreased. Isothermal titration calorimetry (ITC) indicated that polyP binds directly to the MglA/SspA complex with high affinity (KD = 0.3 µM). These observations directly correlated with results obtained from calorimetric scans (DSC), where a strong shift in the mid-transition temperature (Tm) of the MglA/SspA complex was observed in the presence of polyP.