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preface
Microbial cell walls are essential for maintaining cell shape and resisting external stressors. The main structural component of the cell wall is peptidoglycan, a sugar polymer with peptide crosslinks located outside the cell membrane. The biosynthesis and structure of peptidoglycan respond to changing environmental conditions, but the underlying mechanisms of this adaptation are not fully understood.
Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered through cell membranes bound to undecylenyl phosphate, a recoverable lipid carrier. The identification of a family of transmembrane proteins containing DUF368 and DedA as candidate C55-P transloases fills a critical gap in protein knowledge required for biosynthesis of microbial cell surface polymers.
Gram-negative and Gram-positive bacteria lacking homologous DUF368-containing proteins exhibit alkaline-dependent cell wall and viability defects, as well as elevated cell surface C55-P levels. Genetic interactions of pH-dependent synthesis between DUF368-containing proteins and members of the DedA family suggest that the use of C55-P transporters is dynamic and regulated by environmental inputs. Cholera pathogens require C55-P transporter activity to grow and maintain cell shape in the gut.
Phosphorylated undecylenyl lipids as recyclable carrier molecules play an important role in microbial cell surface glycopolymer biogenesis7. During peptidoglycan biosynthesis, glyco-linked pentapeptide subunits of peptidoglycan assembled in the bacterial cytoplasm are covalently linked to C55-P for transmembrane transport.
After MurJ8 moves the carrier-linked peptidoglycan precursor from the inner lobe of the membrane to the outer lobe, cytipeptide is subsequently incorporated into the polymerized peptidoglycan, leaving behind C55 pyrophosphate. Membrane-associated phosphatases including UppP and PAP2 domain proteins PgpB, YbjG, and LpxT7 hydrolyze C55-PP to C55-P, initiating vector cycling.
C55-P may be flipped back to the cytoplasmic surface of the membrane to complete recovery. Preliminary structural studies suggest that UppP also functions as a C55-P transposase, but the protein responsible for C55-P internalization has not been identified. C55-P recovery is a critical step in the biosynthesis of peptidoglycan and other cell surface sugar polymers. Given its extensive and critical role in cell surface maintenance, C55-P recovery is considered an important target for antimicrobial therapies, and natural antibiotics that inhibit this process have been identified.
Vibrio cholerae is a gram-negative pathogen that causes the diarrhoeal disease cholera, and its pH and ion concentrations change drastically as it enters, transports, and leaves the host gastrointestinal tract. Various sensing and signaling networks enable Vibrio cholerae to adapt to changing environments.
Pathogens respond to altered salinity and pH by using sodium power rather than proton power, thereby powering protein secretion and flagellar-dependent motility, and regulating virulence gene expression. The peptidoglycan composition of Vibrio cholerae is thought to be influenced by environmental inputs, but the effect of pH on V. cholerae cell wall biology is unknown.
DUF368 affects the alkaline fitness of Vibrio cholerae
A recent in vivo transposon insertion sequencing screening targeting determinants of intestinal colonization in contemporary Vibrio cholerae clinical isolates identified a number of genes previously unrelated to the pathogenesis of Vibrio cholerae, including several loci whose function is unknown. One of these genes, VCA0040, was chosen for further study, as similar datasets indicate a general need for intestinal colonization of this locus in Vibrio cholerae.
VCA0040 is predicted to be a multichannel inner membrane protein containing a broadly conserved domain DUF368 of unknown function. The predictive structural model of VCA0040 has large putative fractures with domain characteristics of ligand binding or transport activity. V. cholerae strains lacking VCA0040 exhibit stable growth defects accompanied by a pronounced cell shape phenotype, with VCA0040 cells turning into large spheroids. These V. cholerae sphericalis are viable and produce normal rod-shaped daughter cells.
Stationary phase specific factors may trigger shape defects in VCA0040 cells. Exposure of exponentially grown VCA0040 cells to cell-free waste supernatant from quiescent phase cultures rapidly induces spheroid formation. Thermal lability and high molecular weight factors, as well as D amino acids that regulate the composition of stationary phase peptidoglycan, are excluded.
In LB cultures, Vibrio cholerae enters a stable phase accompanied by medium alkalinization. Since minimal shape defects are observed in neutral buffered M9 medium, it is assumed that VCA0040 cells are sensitive to alkalis. Buffering M9 medium to different pH values showed growth defects at pH8 but not at pH6 or pH7, and acidification of cell-free waste supernatants eliminated spheroid-induced phenotypes.
In buffered LB, vca0040 cells exhibited growth and cell shape defects at pH above 8, but not at pH high. The protein VCA0040, containing DUF368, is required for growth under V. cholerae cell shape integrity and alkaline conditions.
Altered peptidoglycan in DUF368 mutants
The DUF368 domain is almost universally conserved throughout the Vibrio family and is present in thousands of other gram-negative, gram-positive, and archaeal species that inhabit a wide range of niches. Heterologous expression of gram-positive or archaeal DUF368 congeners at least partially complements the alkaline growth deficit of V. cholerae cells at least partially. Both congeners showed predicted structural similarity to VCA0040, indicating that protein function containing DUF368 is conserved not only between gram-negative and gram-positive species, but throughout the microbial community.
Since peptidoglycan is required to maintain bacterial cell shape, it is hypothesized that DUF368 function affects the cell wall. VCA0040 mutants have 1.5-2 times less peptidoglycan than wild type, and moderate crosslinking defects accumulate simultaneously with the peptidoglycan precursor UDP-N-acetylmurayl pentapeptide, indicating cross-upstream peptidoglycan biosynthesis defects-linkages.
These phenotypes are present at neutral pH and are exacerbated by exposure to alkaline conditions. Consistent with the conservative view of DUF368 function, S. aureus deficiency SAOUHSC_00846 also has basic growth defects, decreased peptidoglycan count, and decreased UDP-M5 accumulation and crosslinking. From these data, it is concluded that a protein containing DUF368 is conditionally needed to maintain peptidoglycan production and composition.
C55-P circulation is impaired in DUF368 mutants
The other two observations focused on the study of the DUF368 domain on cell wall assembly. While most DUF368-containing proteins are primarily composed of one or two DUF368 domains, some microorganisms encode dual-domain proteins with DUF368-PAP2, PAP2-DUF368, or DUF368-BacA structures.
During spheroid formation of V. cholerae 0040, pgpB is approximately tenfold induced, which encodes C55-PP phosphatase 20. Despite these associations between DUF368 and C55-related processes, the vca0040 mutant showed only a slight increase in the minimum inhibitory concentration of a range of peptidoglycan-targeted antibiotics, possibly because most cell wall-acting compounds are unable to penetrate the gram-negative outer membrane.
MIC screening in Staphylococcus aureus showed that SAOUHSC_00846 mutants were much more sensitive to amfomycin than wild type under alkaline conditions. Amformycin is a Ca2+-dependent lipopeptide antibiotic that specifically binds to C55-P in the outer layer of the cytoplasmic membrane and inhibits its recycling.
Consistent with previous data, amformycin is known to induce UDP-M5 accumulation and reduce peptidoglycan cross-linking in Staphylococcus aureus 24. SAOUHSC_00846 mutants are also moderately sensitive to thinomycin, which inhibits the first directed and C55-P-dependent steps of parietalic acid and peptidoglycan synthesis as well as bacitracin, which binds to C55-PP and prevents the formation of C55-P films on the extracellular surface.
MIC changes in other peptidoglycan targeted molecules are minimal, and to test the function of DUF368 in gram-negative organisms, heterologous expression is heterologous in the E. coli strain lptD421327 that permeates the outer membrane and is sensitive to amformycin SAOUHSC_00846 or vca0040. Expression of either DUF368-containing protein in Lptd4213 E. coli confers amphotericin resistance.
Peptidoglycan composition and antibiotic susceptibility data indirectly indicate impaired C55-P cycling in SAOUHSC_00846 mutants, particularly in C55-P reinternalization. C55 species in membrane lipid extracts from wild-type and SAOUHSC_00846 cultures were quantified.
Amformycin treatment of wild-type Staphylococcus aureus resulted in significant accumulation of C55-P and the gram-positive restriction C55-P precursor C55-OH28. This phenotype replicates in SAOUSHC_00846 Staphylococcus aureus cells, which are grown in alkaline medium but do not replicate or complement the strain under neutral conditions, providing direct evidence that C55-P homeostasis is associated with SAOUSHC_00846.
A base-dependent increase in C55-P in V. cholerae in V. cholerae was also observed. To specifically quantify C55-P in the outer lobes of the cell membrane, fluorescein-conjugated amphotericin was synthesized and used to label live bacterial cells. Ampho-FL-labeled Staphylococcus aureus SAOUHSC_00846 showed increased signaling relative to wild-type bacteria in alkaline medium, indicating conditionally elevated surface C55-P levels in mutants.
These observations suggest that in DUF368-containing protein mutants, the accumulation of alkalinity of surface C55-P causes an increase in compensatory but ultimately insufficient C55-P synthesis, resulting in defects in surface glycopolymer production and impaired growth. These findings are consistent with the model in which the protein containing DUF368 is a C55-P cyclic transporter active under alkaline conditions.
Genetic interaction between DUF368 and DedA
Since C55-P recovery is considered an essential function and DUF368 mutants are conditionally viable, synthetic transposon screening is performed next to define the genetic network of vca0040. Many of the identified synthetic pathological or lethal interactions are associated with cytocapsular homeostasis.
vca0040 interacts most strongly with N900_RS16280, encoding a homologue of YghB, a member of the E. coli DedA family. Vibrio cholerae expresses two other DedA proteins, but these are no hits on this screen. DedA family transmembrane proteins are conserved in all three domains of life, are generally required for cellular envelope homeostasis, and are suspected of mediating PMF-dependent transport, but their specific substrates are unknown.
Although most sequences containing DedA encode only this domain, there are many instances of domain architectures where DedA is fused to the C55-PP phosphatase domain PAP2. The synthetic lethality of vca0040 and yghB was verified by genetic depletion and cross-synthetic transposon screening, and overexpression of YghB rescued the alkaline defect of V. cholerae in vca0040.
Interactions between DUF368 and DedA proteins have also been observed in Staphylococcus aureus. In the alkali-rich spontaneous inhibitors SAOUHSC_00846 Staphylococcus aureus, frequent promoter mutations were found in genes encoding the DedA protein of two Staphylococcus aureus.
Incorporating these mutations into the isopropyl β-D-1-thiogalactopyranoside induction system expressing either DedA protein saved SAOUHSC_00846 even without induction, suggesting that isolated mutations increased gene expression and compensated for the loss of SAOUHSC_00846, such as V. cholerae.
These genetic studies suggest that members of the DedA family are also required for C55-P translocations, consistent with recent reports that eukaryotic DedA proteins are associated with lipid transport and that bacterial DedA mutants are defective in C55 vector-dependent lipopolysaccharide modifications.
The concept of pH-dependent transposase contribution is also supported by the sensitivity of basic amfomycin to lptd4213 E. coli, which has only DedA paralogs and no homologous DUF368-containing protein. Strains lacking yghB and vca0040 survived at acidic pH, suggesting that at least one other Vibrio cholerae protein could undergo C55-P translocation, or that C55-P could spontaneously cross membranes under acidic conditions.
conclusion
Proteins containing the DUF368 and DedA families are proposed to be C55-P translocases, providing functional resilience in this critical step of envelope maintenance. The identification of the C55-P transporter temporarily fills a major gap in the C55 lipid carrier cycling pathway. Since genetic and phenotypic data do not explicitly exclude transport independent activities, biochemical and structural studies are needed to confirm whether these two families actually perform this function.
The apparent pH and ion-dependent contribution of these candidate transloidases to fitness suggests that these proteins can be energetically controlled, as the main effect of pH on microbial cells is the control of transmembrane ion gradients. DUF368-containing proteins appear to contribute most under less alkaline conditions of PMF, and if the C55-P translocation is energy-dependent, DUF368-mediated transport may depend on SMF or other non-PMF energy gradients.
The sensitivity of V. cholerae to increased sodium ion concentrations by Vca0040 and the apparent enrichment of DUF368 in other known halophilic bacteria such as Staphylococcus aureus and archaeal halophiles suggest that DUF368-containing proteins may promote microbial adaptation to high-salt environments, regardless of their energy input.
The sodium and pH-dependent physiology of VCA0040's contribution to Vibrio cholerae may reflect the robust ability of cholera pathogens to colonize the human small intestine, where alkaline conditions and millimolar sodium concentrations may occur. In addition to the variable conservatism of redundant transloidases, additional input and adaptation mechanisms may also influence how the C55-P cycle promotes microbial adaptation.
Although vca0040 is only required under alkaline conditions of Vibrio cholerae, the gene is essential in the associated pathogen Vibrio parahaemolyticus even at neutral pH, although yghB is a conserved and inhibitory locus, indicating that the pH set point where DUF368 family activity dominates is not universal.
Although the protein containing DUF368, which has been renamed polyisoprene phosphate transporter protein in the work reported at the same time, is limited to bacteria and archaea – members of the DedA family are widely present in eukaryotes, and the findings may affect the understanding of polyisoprene phosphate translocations across all kingdoms of life.
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