Novel coordination of lipopolysaccharide modifications in Vibrio cholerae promotes CAMP resistance

In the environment and during infection, the human intestinal pathogen Vibrio cholerae must overcome noxious compounds that damage the bacterial outer membrane. The El Tor and classical biotypes of O1 V. cholerae show striking differences in their resistance to membrane disrupting cationic antimicrobial peptides (CAMPs), such as polymyxins. The classical biotype is susceptible to CAMPs, but current pandemic El Tor biotype isolates gain CAMP resistance by altering the net charge of their cell surface through glycine modification of lipid A. Here we report a second lipid A modification mechanism that only functions in the V. cholerae El Tor biotype. We identify a functional EptA ortholog responsible for the transfer of the amino‐residue phosphoethanolamine (pEtN) to the lipid A of V. cholerae El Tor that is not functional in the classical biotype. We previously reported that mildly acidic growth conditions (pH 5.8) downregulate expression of genes encoding the glycine modification machinery. In this report, growth at pH 5.8 increases expression of eptA with concomitant pEtN modification suggesting coordinated regulation of these LPS modification systems. Similarly, efficient pEtN lipid A substitution is seen in the absence of lipid A glycinylation. We further demonstrate EptA orthologs from non‐cholerae Vibrio species are functional.     

The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica

The PmrA/PmrB regulatory system of Salmonella enterica controls the modification of lipid A with aminoarabinose and phosphoethanolamine. The aminoarabinose modification is required for resistance to the antibiotic polymyxin B, as mutations of the PmrA-activated pbg operon or ugd gene result in strains that lack aminoarabinose in their lipid A molecules and are more susceptible to polymyxin B. Additional PmrA-regulated genes appear to participate in polymyxin B resistance, as pbgP and ugd mutants are not as sensitive to polymyxin B as a pmrA mutant. Moreover, the role that the phosphoethanolamine modification of lipid A plays in the resistance to polymyxin B has remained unknown. Here we address both of these questions by establishing that the PmrA-activated pmrC gene encodes an inner membrane protein that is required for the incorporation of phosphoethanolamine into lipid A and for polymyxin B resistance. The PmrC protein consists of an N-terminal region with five transmembrane domains followed by a large periplasmic region harboring the putative enzymatic domain. A pbgP pmrC double mutant resembled a pmrA mutant both in its lipid A profile and in its susceptibility to polymyxin B, indicating that the PmrA-dependent modification of lipid A with aminoarabinose and phosphoethanolamine is responsible for PmrA-regulated polymyxin B resistance.     

Conflicting roles for a cell surface modification in Salmonella

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O‐antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram‐negative bacteria. We determined that activation of the PmrA/PmrB two‐component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA‐activated gene wzz_st, which encodes the protein that determines long O‐antigen chain length and confers resistance to complement‐mediated killing. By contrast, this growth defect did not require the wzz_fepE gene, which controls production of very long O‐antigen, or other PmrA‐activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O‐antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer‐membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O‐antigen in its LPS to respond to the different conditions it faces during infection.     

pmrA(Con) confers pmrHFIJKL-dependent EGTA and polymyxin resistance on msbB Salmonella by decorating lipid A with phosphoethanolamine

Mutations in pmrA were recombined into Salmonella strain ATCC 14028 msbB to determine if pmrA-regulated modifications of lipopolysaccharide could suppress msbB growth defects. A mutation that functions to constitutively activate pmrA [pmrA(Con)] suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis showed that Salmonella msbB pmrA(Con) strains, compared to Salmonella msbB strains, have increased amounts of palmitate and phosphoethanolamine but no aminoarabinose addition, suggesting that aminoarabinose is not incorporated into msbB lipid A. Surprisingly, loss-of-function mutations in the aminoarabinose biosynthetic genes restored EGTA and polymyxin sensitivity to Salmonella msbB pmrA(Con) strains. These blocks in aminoarabinose biosynthesis also prevented lipid A phosphoethanolamine incorporation and reduced the levels of palmitate addition, indicating previously unknown roles for the aminoarabinose biosynthetic enzymes. Lipid A structural analysis of the EGTA- and polymyxin-resistant triple mutant msbB pmrA(Con) pagP::Tn10, which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on Salmonella msbB strains. Additionally, palmitoylated lipid A was observed only in wild-type Salmonella grown in the presence of salt in rich media. Thus, we correlate EGTA resistance and polymyxin resistance with phosphoethanolamine-decorated lipid A and demonstrate that the aminoarabinose biosynthetic proteins play an essential role in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbB strains.     

Modification of lipid A structure and activity by the introduction of palmitoyltransferase gene to the acyltransferase‐knockout mutant of Escherichia coli

Lauroyltransferase gene (lpxL), Myristoyltransferase gene (lpxM) and palmitoyltransferase gene (crcA) of Escherichia coli BL21 were independently disrupted by the insertional mutations. The knockout mutant of two transferase genes (lpxL and crcA) produced lipid A with no lauric or palmitic acids and only a little amount of myristic acid. The mutant was susceptible to polymyxin B, but showed comparable growth with the wild‐type strain at 30°C. The palmitoyltransferase gene from E. coli (crcA) or Salmonella (pagP) was amplified by PCR, cloned in pUC119, and transferred into the double‐knockout mutant by transformation. The transformant contained palmitic acid in the lipid A, and recovered resistance to polymyxin B. Mass spectrometric analysis revealed that palmitic acid was linked to the hydroxyl group of 3‐hydroxymyristic acid at C‐2 position of proximal (reducing‐end) glucosamine. LPS from the double‐knockout mutant showed reduced IL‐6‐inducing activity to macrophage‐like line cells compared to that of the wild‐type strain, and the activity was only slightly restored by the introduction of palmitic acid to the lipid A. These results suggested that the introduction of one palmitic acid was enough to recover the integrity of the outer membrane, but not enough for the stimulation of macrophages.

     

Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ‐dependent 4‐amino‐4‐deoxy‐l‐arabinose addition to lipid A

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare‐associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram‐negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine‐containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two‐component system (TCS) directly activates 4‐amino‐4‐deoxy‐l ‐arabinose (l ‐Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l ‐Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrAB_Ecl TCS. Instead, PhoP_Ecl binds to the arnB_Ecl promoter to induce l ‐Ara4N biosynthesis and PmrAB‐independent addition to the lipid A disaccharolipid. Therefore, PhoPQ_Ecl contributes to regulation of CAMP heteroresistance in some ECC clusters.     

An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor

Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium. The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified. We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors. No soluble factors are required. A gene located near minute 51 on the S. typhimurium and E. coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase. The enzyme, renamed ArnT, consists of 548 amino acid residues in S. typhimurium with 12 possible membrane-spanning regions. ArnT displays distant similarity to yeast protein mannosyltransferases. ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide. However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo. Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis. As shown in the following article (Trent, M. S., Ribeiro, A. A., Doerrler, W. T., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2001) J. Biol. Chem. 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.     

Long tail fibres of the novel broad‐host‐range T‐even bacteriophage S16 specifically recognize Salmonella OmpC

We report isolation and characterization of the novel T4‐like Salmonella bacteriophage vB_SenM‐S16. S16 features a T‐even morphology and a highly modified 160 kbp dsDNA genome with 36.9 mol % G+C, containing 269 putative coding sequences and three tRNA genes. S16 is a virulent phage, and exhibits a maximally broad host range within the genus Salmonella, but does not infect other bacteria. Synthesis of functional S16 full‐length long tail fibre (LTF) in Escherichia coli was possible by coexpression of gp37 and gp38. Surface plasmon resonance analysis revealed nanomolar equilibrium affinity of the LTF to its receptor on Salmonella cells. We show that OmpC serves as primary binding ligand, and that S16 adsorption can be transferred to E. coli by substitution of ompC with the Salmonella homologue. S16 also infects ‘rough’ Salmonella strains which are defective in lipopolysaccharide synthesis and/or its carbohydrate substitution, indicating that this interaction does not require an intact LPS structure. Altogether, its virulent nature, broad host range and apparent lack of host DNA transduction render S16 highly suitable for biocontrol of Salmonella in foods and animal production. The S16 LTF represents a highly specific affinity reagent useful for cell decoration and labelling, as well as bacterial immobilization and separation.     

The Role of Oxidoreductases in Determining the Function of the Neisserial Lipid A Phosphoethanolamine Transferase Required for Resistance to Polymyxin

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.     

Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium

The covalent addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid A, which resides in the outer membranes of bacteria such as Salmonella typhimurium and Escherichia coli, is the final step in the polymyxin-resistance pathway in these organisms. This modification is catalyzed by the inner membrane protein 4-amino-4-deoxy-L-arabinose transferase (ArnT). Little is known about the ArnT protein structure because it has not previously been purified. We report here the first expression and purification of 6 x His-tagged S. typhimurium ArnT in NovaBlue cells. The enzyme was purified using sequential Q-Sepharose anion exchange and HisLink nickel affinity column chromatography. The purified protein has an apparent molecular weight of 62 kDa on SDS-PAGE and the identity of the purified ArnT was confirmed by Western blot using a monoclonal antibody against the His-tag and by MALDI-TOF mass spectrometry. Purified ArnT protein was shown to be highly alpha-helical as determined by circular dichroism analysis. A chromosomal ArnT knockout strain of E. coli BL21(DE3) was developed to allow in vivo functional analysis of plasmid-encoded ArnT constructs, and a polymyxin assay was used to confirm that the cloned ArnT proteins retained full activity. These studies provide an essential foundation for further analysis of ArnT structure and function using mutagenesis and biophysical techniques.     

Temperature-dependent regulation of the Escherichia coli lpxT gene

The Lipid A moiety of the lipopolysaccharide can be covalently modified during its transport to the outer membrane by different enzymes, among which the LpxT inner membrane protein. LpxT transfers a phosphate group from the undecaprenyl pyrophosphate to the Lipid A, a modification affecting the stability of the outer membrane and its recognition by the host immune system in Enterobacteria. We previously found that the expression of the Pseudomonas aeruginosa lpxT gene, encoding LpxT, is induced in response to a temperature upshift and we proposed that an RNA thermometer was responsible for such regulation. Here we show that the Escherichia coli lpxT orthologous gene is down-regulated upon a temperature upshift and investigated the mechanism of this regulation. We found that the LpxT protein stability is not affected by the temperature change. Conversely, the lpxT mRNA levels strongly decrease upon a shift from 28 to 42 °C. The lack of MicA sRNA, which was previously implicated in lpxT regulation, does not affect lpxT thermal regulation. We identified the lpxTp promoter and demonstrated that lpxTp has temperature-sensitive activity depending on its peculiar −10 region. Moreover, we found that RNase E-dependent degradation of the lpxT mRNA is also modulated by temperature causing a strong destabilization of the lpxT mRNA at 42 °C. In vitro data argue against the involvement of factors differentially expressed at 28 and 42 °C in the temperature–dependent modulation of lpxT mRNA stability.     

Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(III) and Al(III)

Iron is an essential metal but can be toxic in excess. While several homeostatic mechanisms prevent oxygen-dependent killing promoted by Fe(II), little is known about how cells cope with Fe(III), which kills by oxygen-independent means. Several Gram-negative bacterial species harbour a regulatory system - termed PmrA/PmrB - that is activated by and required for resistance to Fe(III). We now report the identification of the PmrA-regulated determinants mediating resistance to Fe(III) and Al(III) in Salmonella enterica serovar Typhimurium. We establish that these determinants remodel two regions of the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane. Remodelling entails the covalent modification of the two phosphates in the lipid A region with phosphoethanolamine and 4-aminoarabinose, which has been previously implicated in resistance to polymyxin B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant lacking the PmrA-regulated Fe(III) resistance genes bound more Fe(III) than the wild-type strain and was defective for survival in soil, suggesting that these PmrA-regulated lipopolysaccharide modifications aid Salmonella's survival and spread in non-host environments.     

Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene

Fluorescent reference strains of bacteria carrying a stable chromosomally integrated single copy of the gfp gene have been developed. A modified version of the gfp gene has been generated by mutagenesis and expressed under the control of the bacteriophage lambda promoter P_L. A cassette comprising bacteriophage Mu transposon arms flanking the modified gfp gene and regulatory regions was irreversibly integrated as an in-vitro-assembled transposition complex into the genomes of Escherichia coli and Salmonella spp. The modified green fluorescent protein (GFP) protein retained the fluorescence excitation and emission wavelengths of wild-type GFP. However, it fluoresced more brightly in E. coli and Salmonella compared to wild-type GFP, presumably due to improved protein maturation. Fluorescent E. coli and Salmonella strains carrying the gfp gene cassette were easily differentiated from their respective non-fluorescent parental strains on various growth media by visualization under UV light. The bacterial strains produced by this method remained viable and stably fluorescent when incorporated into a matrix for delivery of exact numbers of viable bacterial cells for use as quality control agents in microbiological procedures.     

Salmonella modulation of the phagosome membrane,role of SseJ

Salmonellae have the ability to invade, persist and replicate within an intracellular phagosome termed the Salmonella‐containing vacuole (SCV). Salmonellae alter lipid and protein content of the SCV membrane and manipulate cytoskeletal elements in contact with the SCV using the Salmonella pathogenicity island 1 (SPI‐2) type III secretion system effectors. These modifications result in microtubular‐based movement and morphological changes, which include endosomal tubulation of the SCV membrane. SseJ is a SPI‐2 effector that localizes to the cytoplasmic face of the SCV and esterifies cholesterol through its glycerophospholipid : cholesterol acyltransferase activity. SseJ enzymatic activity as well as localization to the SCV are determined by binding to the small mammalian GTPase, RhoA. This review will focus on current knowledge about the role of SseJ in SCV membrane modification and will discuss how the hypothesis that a major role of SPI‐2 effectors is to modify SCV protein and lipid content to promote bacterial intracellular survival.