Cristina L. Marolda

Intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae.

Members of the taxonomically diverse Burkholderia cepacia complex have become a major health risk for patients with cystic fibrosis (CF). Although patient-to-patient transmission of B. cepacia strains has been well-documented, very little is known about possible vehicles of transmission and reservoirs for these micro-organisms. In this work, it is shown that strains of the B. cepacia complex can survive within different isolates of the genus Acanthamoeba. Trophozoites containing bacteria developed profuse cytoplasmic vacuolization. Vacuolization was not detected in trophozoites infected with live Escherichia coli or heat-killed B. cepacia, or by incubation of trophozoites with filter-sterilized culture supernatants, indicating that metabolically active intracellular bacteria are required for the formation of vacuoles. Experiments with two different B. cepacia strains and two different Acanthamoeba isolates revealed that bacteria display a low level of intracellular replication approximately 72-96 h following infection. In contrast, extracellular bacteria multiplied efficiently on by-products released by amoebae. The findings suggest that amoebae may be a reservoir for B. cepacia and possibly a vehicle for transmission of this opportunistic pathogen among CF patients.

Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid‐linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni‐encoded ABC‐type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate‐linked heptasaccharide intermediate involved in the recently identified bacterial N‐linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N‐linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O‐antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.

Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria (28). It has a tripartite structural organization consisting of lipid A, a conserved core oligosaccharide region, and an O-specific polysaccharide chain or O antigen. In the majority of gram-negative bacteria, the core oligosaccharide can be subdivided into an outer core, generally composed of hexoses and hexosamines, and an inner core made of 3-deoxy-d-manno-oct-2-ulosonic acid and l,d-heptose units. LPS plays an important role in maintaining the structural integrity of the bacterial outer membrane by interacting with outer membrane proteins and divalent cations (15), thereby providing a barrier against the entry of toxic hydrophobic compounds into the bacterial cell (27). Escherichia coli mutants defective in the biosynthesis of 3-deoxy-d-manno-oct-2-ulosonic acid are nonviable, whereas those impaired in l,d-heptose synthesis survive in vitro, although they display a pleiotropic phenotype referred to as “deep rough” (17). This phenotype is characterized by an extreme sensitivity to very low concentrations of novobiocin, detergents, and bile salts (32). Deep rough mutants also have defects in F plasmid conjugation and generalized transduction by the bacteriophage P1 (6, 16). Haemophilus influenzae heptose-deficient mutants were found to be serum sensitive and displayed a reduced virulence in vivo (18, 36). The complete biosynthesis pathway of the l,d-heptose precursor has not been elucidated. Eidels and Osborn (11) proposed a four-step pathway for the synthesis of NDP-l,d-heptose, which is still widely accepted in the literature (see reference 13 for a review). It includes (i) conversion of d-sedoheptulose 7-phosphate to d,d-heptose 7-phosphate by a phosphoheptose isomerase; (ii) formation of d,d-heptose 1-phosphate by a phosphoheptose mutase; (iii) activation of the d,d-heptose 1-phosphate intermediate to NDP-d,d-heptose by an NDP-heptose synthetase; and (iv) epimerization of the NDP-heptose to form the final product, NDP-l,d-heptose. Subsequent studies involving the isolation of ADP-d,d-heptose and ADP-l,d-heptose from Shigella sonnei and Salmonella enterica serovar Typhimurium indicated that ADP is the activating nucleotide (20–22). In the absence of purified ADP-heptose, Kadrmas and Raetz (19) used ADP-mannose as a substrate for the E. coli heptosyltransferase I (WaaC). More recently, it has been clearly demonstrated that heptosyltransferases I and II (WaaF) from E. coli accept ADP-l-β-d-heptose and ADP-d-β-d-heptose as substrates, although the efficiency of the transfer reactions with the d-β-d isomer is markedly reduced (14, 35). In gram-negative bacteria, functional studies have only been performed for the isomerization reaction and the epimerization step (3, 9, 26), while the conversion of d,d-heptose 7-phosphate to d,d-heptose 1-phosphate and a functional proof of the activating step have not been demonstrated. The d-sedoheptulose 7-phosphate isomerase activity was described in S. enterica serovar Typhimurium (12), and the corresponding gene, gmhA, has been cloned both from E. coli and from H. influenzae (3, 4). The amino acid sequence of the GmhA polypeptide is highly conserved in different gram-negative bacteria (33). The epimerization step is catalyzed by the WaaD (formerly RfaD) protein (5), which has also been crystallized (8). We have recently shown that the E. coli rfaE gene product consists of two distinct domains that may be involved in the biosynthesis of d,d-heptose 1-phosphate, as well as the activating step (34). It was demonstrated that one of the RfaE domains shares structural features with members of the ribokinase family, while the other domain has conserved features present in nucleotidyltransferases (34). The demonstration of a protein domain corresponding to a putative sugar kinase suggested that the original pathway for NDP-heptose biosynthesis as proposed by Eidels and Osborn may not be accurate and, at the same time, predicted the existence of an additional phosphatase step (33). The complete biosynthesis pathway of GDP-d-α-d-heptose from d-sedoheptulose 7-phosphate in the gram-positive bacterium Aneurinibacillus thermoaerophilus DSM 10155 was recently characterized (20). We demonstrated that two independent enzymes catalyze the originally proposed mutase step. A d,d-heptose 7-phosphate kinase adds a phosphate group at the C-1 position, and subsequently a d,d-heptose 1,7-bisphosphate phosphatase removes the phosphate group at the C-7 position. The GDP-activated d,d-isomer serves as a precursor for the incorporation of the heptose into the glycan moiety of a surface layer (S-layer) glycoprotein produced by A. thermoaerophilus (20). Amino acid sequence analysis of completely sequenced genomes revealed that the A. thermoaerophilus phosphatase is highly conserved among different gram-negative bacteria (20), in agreement with a previous suggestion that a phosphatase reaction is also required for the synthesis of ADP-d,d-heptose and ADP-l,d-heptose in these microorganisms (34). In the present study, we report the reconstruction in vitro with purified enzyme components of the complete biosynthesis pathway for ADP-d-β-d-heptose in E. coli. We also provide genetic evidence demonstrating that the function of a novel phosphatase gene in E. coli K-12, now designated gmhB (formerly yaeD), is required for the synthesis of ADP-d-β-d-heptose. Furthermore, we propose a new gene nomenclature to account for the differences and similarities between the components of the pathways leading to the formation of ADP-l-β-d-heptose and GDP-d-α-d-heptose in gram-negative and gram-positive bacteria, respectively.

Interplay of the Wzx Translocase and the Corresponding Polymerase and Chain Length Regulator Proteins in the Translocation and Periplasmic Assembly of Lipopolysaccharide O Antigen

Genetic evidence suggests that a family of bacterial and eukaryotic integral membrane proteins (referred to as Wzx and Rft1, respectively) mediates the transbilayer movement of isoprenoid lipid-linked glycans. Recent work in our laboratory has shown that Wzx proteins involved in O-antigen lipopolysaccharide (LPS) assembly have relaxed specificity for the carbohydrate structure of the O-antigen subunit. Furthermore, the proximal sugar bound to the isoprenoid lipid carrier, undecaprenyl-phosphate (Und-P), is the minimal structure required for translocation. In Escherichia coli K-12, N-acetylglucosamine (GlcNAc) is the proximal sugar of the O16 and enterobacterial common antigen (ECA) subunits. Both O16 and ECA systems have their respective translocases, WzxO16 and WzxE, and also corresponding polymerases (WzyO16 and WzyE) and O-antigen chain-length regulators (WzzO16 and WzzE), respectively. In this study, we show that the E. coli wzxE gene can fully complement a wzxO16 translocase deletion mutant only if the majority of the ECA gene cluster is deleted. In addition, we demonstrate that introduction of plasmids expressing either the WzyE polymerase or the WzzE chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of WzxE. We also show that this property is not unique to WzxE, since WzxO16 and WzxO7 can cross-complement translocase defects in the O16 and O7 antigen clusters only in the absence of their corresponding Wzz and Wzy proteins. These genetic data are consistent with the notion that the translocation of O-antigen and ECA subunits across the plasma membrane and the subsequent assembly of periplasmic O-antigen and ECA Und-PP-linked polymers depend on interactions among Wzx, Wzz, and Wzy, which presumably form a multiprotein complex.

Surface expression of O-specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein.

We investigated the involvement of Tol proteins in the surface expression of lipopolysaccharide (LPS). tolQ, ‐R, ‐A and ‐B mutants of Escherichia coli K‐12, which do not form a complete LPS‐containing O antigen, were transformed with the O7+ cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7‐deficient phenotype in the tolQ and tolA mutants was complemented with a plasmid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a direct mutation of this gene or by a polar effect on tolA gene expression exerted by the tolQ mutation. Reduced surface expression of O7 LPS was not caused by changes in lipid A‐core structure or downregulation of the O7 LPS promoter. However, an abnormal accumulation of radiolabelled mannose was detected in the plasma membrane. As mannose is a sugar unique to the O7 subunit, this result suggested the presence of accumulated O7 LPS biosynthesis intermediates. Attempts to construct a tolA mutant in the E. coli O7 wild‐type strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 caused slow growth rate and serum sensitivity in addition to reduced O7 LPS production. VW187 tolQ cells showed an elongated morphology and became permeable to the membrane‐impermeable dye propidium iodide. All these phenotypes were corrected upon complementation with cloned tol genes but were not restored by complementation with the tolQRA operon containing the frameshift mutation in tolA. Our results demonstrate that the TolA protein plays a critical role in the surface expression of O antigen subunits by an as yet uncharacterized involvement in the processing of O antigen.

Staphylococcus aureus Transporters Hts, Sir, and Sst Capture Iron Liberated from Human Transferrin by Staphyloferrin A, Staphyloferrin B, and Catecholamine Stress Hormones, Respectively, and Contribute to Virulence

ABSTRACT Staphylococcus aureus is a frequent cause of bloodstream, respiratory tract, and skin and soft tissue infections. In the bloodstream, the iron-binding glycoprotein transferrin circulates to provide iron to cells throughout the body, but its iron-binding properties make it an important component of innate immunity. It is well established that siderophores, with their high affinity for iron, in many instances can remove iron from transferrin as a means to promote proliferation of bacterial pathogens. It is also established that catecholamine hormones can interfere with the iron-binding properties of transferrin, thus allowing infectious bacteria access to this iron pool. The present study demonstrates that S. aureus can use either of two carboxylate-type siderophores, staphyloferrin A and staphyloferrin B, via the transporters Hts and Sir, respectively, to access the transferrin iron pool. Growth of staphyloferrin-producing S. aureus in serum or in the presence of holotransferrin was not enhanced in the presence of catecholamines. However, catecholamines significantly enhanced the growth of staphyloferrin-deficient S. aureus in human serum or in the presence of human holotransferrin. It was further demonstrated that the Sst transporter was essential for this activity as well as for the utilization of bacterial catechol siderophores. The substrate binding protein SstD was shown to interact with ferrated catecholamines and catechol siderophores, with low to submicromolar affinities. Experiments involving mice challenged intravenously with wild-type S. aureus and isogenic mutants demonstrated that the combination of Hts, Sir, and Sst transport systems was required for full virulence of S. aureus.

Micromethods for the Characterization of Lipid A-Core and O-Antigen Lipopolysaccharide

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.

Defective O-Antigen Polymerization in tolA and pal Mutants of Escherichia coli in Response to Extracytoplasmic Stress

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1)

In previous studies the authors cloned and characterized the DNA sequence of the regions at both ends of the O7-specific lipopolysaccharide (LPS) biosynthesis cluster of Escherichia coli VW187 (O7:K1), and identified the biosynthetic genes for dTDP-rhamnose and GDP-mannose, as well as one of the candidate glycosyltransferases. In this work the complete DNA sequence of a 6.9 kb intervening region is presented. Seven new ORFs were identified. All the functions required for the synthesis and transfer of the O7 LPS were assigned on the basis of complementation experiments of transposon insertion mutants, and amino acid sequence homology to proteins involved in LPS synthesis of other bacteria. Of the seven ORFs, two encoded membrane proteins that were homologous to the O-antigen translocase (Wzx) and polymerase (Wxy), two were involved in the biosynthesis of dTDP-N-acetylviosamine, and the remaining three showed homologies to sugar transferases. The O antigen chain length regulator gene wzz was also identified in the vicinity of the O7 polysaccharide cluster. O7-specific DNA primers were designed and tested for serotyping of O7 E. coli strains.

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in DeltawbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the DeltawbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.