Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis. Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the essential flippase that translocates it across the cytoplasmic membrane for PG polymerization is unclear. We developed an assay for lipid II flippase activity and used a chemical genetic strategy to rapidly and specifically block flippase function. We combined these approaches to demonstrate that MurJ is the lipid II flippase in Escherichia coli.

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.T he cell envelope of most bacteria contains a cell wall exoskeleton composed of peptidoglycan (PG) that surrounds the cytoplasmic membrane (1, 2). The rigid PG structure protects the bacterium from osmotic rupture, serves as a scaffold onto which other envelope components are attached, and defines cell shape. Underscoring the essentiality of the PG cell wall is the fact that many antibiotics target PG biosynthesis (3).Bacteria build their PG sacculus by polymerizing an N-acetylglucosamine-N-acetylmuramic acid (GlcNAc-MurNAc) disaccharide-pentapeptide into long glycan chains that are cross-linked by peptide bonds between stem peptides (2). This GlcNAcMurNAc disaccharide-pentapeptide is synthesized at the cytoplasmic side of the membrane as a polyisoprenyl lipid-linked precursor known as lipid II (Fig. 1A) (4). Because lipid II polymerization occurs at the extracytoplasmic side of the membrane, an obligatory step in PG biosynthesis is the translocation of the lipidlinked disaccharide-pentapeptide across the cytoplasmic membrane.The use of polyisoprenyl lipid-linked precursors in the biogenesis of envelope glycopolymers is widespread in bacteria. Examples include the biogenesis of PG, certain capsules and exopolysaccharides, and O antigens (5, 6). In these systems, bacteria build each precursor ...

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogs (NAs) suppresses hepatitis B virus (HBV) DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). Hepatitis B virus pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual-target real-time quantitative PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. Hepatitis B virus DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV-positive populations. Samples included on-therapy CHB samples (n = 16), samples (n = 89) from 10 treatment naïve CHB subjects receiving 12 weeks of NA treatment with 8-week follow-up, hepatitis B surface antigen-positive blood donor samples (n = 102), and three seroconversion series from plasmapheresis donors (n = 79 samples). Conclusion: During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions.

Staphylococcus aureus encodes the Sec-independent Ess secretion pathway, an ortholog of mycobacterial T7 secretion systems which is required for the virulence of this Gram-positive microbe. The Ess (ESX secretion) pathway was previously defined as a genomic cluster of eight genes, esxA, esaA, essA, essB, esaB, essC, esaC, and esxB. essABC encode membrane proteins involved in the stable expression of esxA, esxB, and esaC, genes specifying three secreted polypeptide substrates. esaB, which encodes a small cytoplasmic protein, represses the synthesis of EsaC but not that of EsxA and EsxB. Here we investigated a hitherto uncharacterized gene, esaD, located downstream of esxB. Expression of esaD is activated by mutations in esaB and essB. EsaD, the 617-amino-acid product of esaD, is positioned in the membrane and is also accessible to EsaD-specific antibodies on the bacterial surface. S. aureus mutants lacking esaD are defective in the secretion of EsxA. Following intravenous inoculation of mice, S. aureus esaD mutants generate fewer abscesses with a reduced bacterial load compared to wild-type parent strain Newman. The chromosomes of Listeria and Bacillus species with Ess pathways also harbor esaD homologues downstream of esxB, suggesting that the contributory role of EsaD in Ess secretion may be shared among Gram-positive pathogens.

Proteins of the WXG100 family represent the prototypical substrates of bacterial type VII secretion systems that typically encompass 100 residues, lack canonical signal peptides, and form helix-turn-helix hairpin structures with WXG positioned in the turn element. Bacillus anthracis encodes six WXG100 proteins, herein referred to as EsxB, EsxL, EsxP, EsxQ, EsxV, and EsxW. With the exception of EsxB, B. anthracis proteins harbor C-terminal extensions that are appended to canonical WXG domains. When cultured in liquid broth, B. anthracis secretes two substrates, EsxB and EsxW, into the extracellular environment. EsxB is required for the stability and secretion of EsxW; however, EsxW is dispensable for EsxB secretion. In agreement with the hypothesis that EsxB binding to substrates promotes recognition and secretion by the type VII pathway, EsxB is reported to interact with EsxB and EsxW. Unlike deletions in mycobacterial EsxB, deletion of five N-or C-terminal residues does not affect the ability of mutant B. anthracis EsxB to travel the type VII pathway and initiate secretion of EsxW. Translational fusion of ubiquitin to the N or C terminus of EsxB also had no effect, while ubiquitin insertion into the center turn abrogated secretion. Anthrax-infected guinea pigs mounted humoral immune responses to EsxB, EsxP, and EsxW, which suggests that B. anthracis activates the type VII secretion pathway during infection.

Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport. Most bacteria produce a rigid peptidoglycan (PG) layer that is essential for defining cell shape and providing protection against osmotic lysis (1, 2). This mesh-like PG macromolecule consists of glycan strands of repeating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide units that are bridged by the cross-linking of peptide stems extending from the MurNAc moiety. The PG layer is localized outside the cytoplasmic membrane, and as the cell grows, newly synthesized PG material must be incorporated into the preexisting polymer. Since the disaccharide-pentapeptide building blocks are made in the cytoplasm, the cytoplasmic membrane separates PG precursor synthesis from its utilization. Therefore, transport of the disaccharide-pentapeptide across the membrane is an essential step in PG biogenesis.In Escherichia coli, PG is present in the periplasm, the aqueous compartment between the inner and outer membranes (3). Like in all PG-producing bacteria, the disaccharide-pentapeptide PG precursor is synthesized onto the lipid carrier undecaprenol at the inner leaflet of the inner membrane (IM) (reviewed in references 4-7). This lipid-linked precursor, known as lipid II (undecaprenyl-pyrophosphoryl-MurNAc-[pentapeptide]-GlcNAc), must be flipped across the IM and delivered to the outer leaflet of the IM. Once lipid II is exposed to the periplasm, t...

Infection and disease associated with Neisseria gonorrhoeae, the gonococcus, continue to be a global health problem. Asymptomatic and subclinical gonococcal infections occur at a high frequency in females; thus, the true incidence of N. gonorrhoeae infections are presumed to be severely underestimated. Inherent to this asymptomatic/subclinical diseased state is the continued prevalence of this organism within the general population, as well as the medical, economic, and social burden equated with the observed chronic, disease sequelae. As infections of the lower female genital tract (i.e., the uterine cervix) commonly result in subclinical disease, it follows that the pathobiology of cervical gonorrhea would differ from that observed for other sites of infection. In this regard, the potential responses to infection that are generated by the female reproductive tract mucosa are unique in that they are governed, in part, by cyclic fluctuations in steroid hormone levels. The lower female genital tract has the further distinction of being able to functionally discriminate between resident commensal microbiota and transient pathogens. The expression of functionally active complement receptor 3 by the lower, but not the upper, female genital tract mucosa; together with data indicating that gonococcal adherence to and invasion of primary cervical epithelial cells and tissue are predominately aided by this surface-expressed host molecule; provide one explanation for asymptomatic/subclinical gonococcal cervicitis. However, co-evolution of the gonococcus with its sole human host has endowed this organism with variable survival strategies that not only aid these bacteria in successfully evasion of immune detection and function but also enhance cervical colonization and cellular invasion. To this end, we herein summarize current knowledge pertaining to the pathobiology of gonococcal infection of the human cervix.

Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), infects an estimated 15–20 million people worldwide and confers a greater risk for accelerated progression to liver disease. However, limited HDV surveillance data are available in sub-Saharan Africa where HDV diversity is high. To determine the prevalence and diversity of HDV in Cameroon, serological and molecular characterization was performed on 1928 HBsAg positive specimens selected from retrospective viral surveillance studies conducted in Cameroon from 2010–2016. Samples were screened for HDV antibodies on the Abbott ARCHITECT instrument and for HDV RNA on the Abbott m2000 instrument by research assays. HDV positive specimens with sufficient viral load were selected for genomic sequencing. The seroprevalence of HDV in HBsAg positive samples from Cameroon was 46.73% [95% CI; 44.51–48.96%], with prevalence of active HDV infection being 34.2% [95% CI; 32.09–36.41%]. HDV genotypes 1, 6, 7 and 8 were identified amongst N = 211 sequences, including N = 145 genomes. HDV prevalence is high within the study cohort, indicating that a large portion of HBV infected individuals in Cameroon are at elevated risk for severe hepatitis and death. Collectively, these results emphasize the need for HBV vaccination and HDV testing in HBsAg positive patients in Cameroon.