Linda van Dijk

Analysis of the F Antigen-Specific papA Alleles of Extraintestinal Pathogenic Escherichia coli Using a Novel Multiplex PCR-Based Assay

ABSTRACT Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenicEscherichia coli, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants ofpapA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of thepapA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence thatpapA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer ofpapA alleles between lineages. Sequencing ofpapA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papA alleles available to any laboratory with PCR capability.

The Campylobacter jejuni PhosS/PhosR operon represents a non-classical phosphate-sensitive two-component system

The bacterial pathogen Campylobacter jejuni carries several putative two‐component signal transduction systems of unknown function. Here we report that the PhosS (Cj0889) and PhosR (Cj0890) proteins constitute a two‐component system that is activated by phosphate limitation. Microarray analysis, real‐time RT‐PCR, and primer extension experiments indicated that this system regulates 12 genes (including the pstSCAB genes) present in three transcriptional units. Gel shift assays confirmed that recombinant PhosR protein bound DNA fragments containing the promoter regions upstream of these three transcriptional units. Although functionally similar, the PhosS/PhosR does not exhibit sequence homology with the classical PhoBR systems, has a different pho box (5′‐GTTTCNAAAANGTTTC‐3′) recognized by the C. jejuni response regulator, and is not autoregulated. Because of these atypical properties, we designated the Cj0889‐Cj0890 operon as the C. jejuni PhosS/PhosR system (phosphate sensor/phosphate response regulator) and the phosphate‐regulated genes as the pho regulon of C. jejuni.

Temperature‐dependent FlgM/FliA complex formation regulates Campylobacter jejuni flagella length

Regulation of the biosynthesis of the flagellar filament in bacteria containing multiple flagellin genes is not well understood. The major food‐borne pathogen Campylobacter jejuni possesses on both poles a flagellum that consists of two different flagellin subunits, FlaA and FlaB. Here we identify the protein Cj1464 as a regulator of C. jejuni flagellin biosynthesis. The protein shares characteristics of the FlgM family of anti‐σ factor proteins: it represses transcription of σ28‐dependent genes, forms a complex with σ factor FliA, and is secreted through the flagellar filament. However, unlike other FlgM proteins, the interaction of C. jejuni FlgM with FliA is regulated by temperature and the protein does not inhibit FliA activity during the formation of the hook‐basal body complex (HBB). Instead, C. jejuni FlgM limits the length of the flagellar filament by suppressing the synthesis of both the σ28‐ and the σ54‐dependent flagellins. The main function of the C. jejuni FlgM therefore is not to silence σ28‐dependent genes until the HBB is completed, but to prevent unlimited elongation of the flagellum, which otherwise leads to reduced bacterial motility.

Genotyping by PCR, of Staphylococcus aureus strains, isolated from mammary glands of cows

A total of 71 Staphylococcus aureus strains isolated from bovine mammary glands were identified and subtyped. The methods used to differentiate between the S. aureus isolates were the DNA polymorphism pattern after amplification with a Polymerase Chain Reaction using several primer combinations and phage typing. The DNA fingerprinting technique using RAPD, ERIC1R and ERIC primers proved to be useful in differentiating isolates of S. aureus. Differentiation of isolates using phage typing gave no additional information compared to the DNA technique. The outbreak of S. aureus in the herd studied was mainly caused by one S. aureus strain. Other strains were only found on three occasions, twice in subclinical infections and once from a case of clinical mastitis. In the latter case the dominant strain was isolated from a different quarter of the same cow. Four of the ten cows studied suffered from clinical mastitis. From those four cows, three remained infected with the same S. aureus strain despite antibiotic treatment.

Antigenic variation within the subunit protein of members of the colonization factor antigen I group of fimbrial proteins in human enterotoxigenic Escherichia coli

Colonization factor antigens (CFAs) of human enterotoxigenic Escherichia coli can be divided in groups based on the N-terminal amino acid sequence of their major subunit protein. One of the groups that has been distinguished in this way, is the CFA/I group of fimbriae. The sequence of the fimbrial subunit genes in the operons encoding the antigens CS4, CS14 and CS17, all members of this group, was determined. A duplication of the fimbrial subunit gene (csuA) was found in the CS14 operon, both genes encoding very similar proteins. Purified CS14 fimbriae consist of two proteins with different molecular masses (15.5 and 17.0 kDa) but identical N-terminal amino acid sequences, which strongly suggests that both csuA genes are transcribed. A phylogenetic tree derived from the amino acid sequences of the CFA/I, CS1, CS2, CS4, CS14, CS17 and CS19 subunit proteins shows that CS1, CS17 and CS19 belong to the same subgroup. CFA/I, CS4 and CS14 belong to a second subgroup, while CS2 is distinct within the CFA/I group of fimbriae. The genetic similarity between CS1, CS17 and CS19 is reflected in the substantial immunological cross-reactivity observed, both between their protein subunits and intact fimbriae.

Isolation and characterisation of dog uropathogenic Proteus mirabilis strains

Proteus mirabilis strains isolated from the urine of dogs with urinary tract infections, were characterised with respect to the production of haemolysin and fimbriae. In contrast to healthy dogs, P. mirabilis was also isolated in high numbers from the faeces of dogs suffering from recurrent urinary tract infections. Production of fimbriae was demonstrated by electron microscopy and the presence of genes for two different types of major fimbrial subunits (MR/P-like or UCA-like) was demonstrated by Southern hybridisation. These genes were absent in the Proteus vulgaris, Providentia rettgeri and Morganella morganii strains tested. All but one P. mirabilis strains were haemolytic and most strains produced fimbriae albeit in different amounts. The UCA fimbrial subunits from dog and human isolates have identical molecular weights and N-terminal sequences and are immunologically cross reactive. It was concluded that dog uropathogenic P. mirabilis strains are very similar to human uropathogenic P. mirabilis strains.

Generation of the membrane potential and its impact on the motility, ATP production and growth in Campylobacter jejuni

The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate‐level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm‐blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.

Function and Regulation of the C4-Dicarboxylate Transporters in Campylobacter jejuni

C4-dicarboxylates are important molecules for the human pathogen C.jejuni, as they are used as carbon and electron acceptor molecules, as sugars cannot be utilized by this microaerophilic organism. Based on the genome analysis, C. jejuni may possess five different C4–dicarboxylate transporters: DctA, DcuA, DcuB, and two homologs of DcuC. Here, we investigated the regulation and function of various C4–dicarboxylate transporters in C. jejuni. Transcription of the dctA and dcuC homologs is constitutive, while dcuA and dcuB are both directly regulated by the two-component RacR/RacS system in response to limited oxygen availability and the presence of nitrate. The DctA transporter is the only C4-dicarboxylate transporter to allow C. jejuni to grow on C4-carbon sources such as aspartate, fumarate, and succinate at high oxygen levels (10% O2) and is indispensable for the uptake of succinate from the medium under these conditions. Both DcuA and DcuB can sequester aspartate from the medium under low-oxygen conditions (0.3% O2). However, under these conditions, DcuB is the only transporter to secrete succinate to the environment. Under low-oxygen conditions, nitrate prevents the secretion of succinate to the environment and was able to overrule the phenotype of the C4-transporter mutants, indicating that the activity of the aspartate–fumarate–succinate pathway in C. jejuni is strongly reduced by the addition of nitrate in the medium.

Regulation of Energy Metabolism by the Extracytoplasmic function (ECF) σ factors of Arcobacter butzleri

The extracytoplasmic function (ECF) σ factors are fundamental for bacterial adaptation to distinct environments and for survival under different stress conditions. The emerging pathogen Arcobacter butzleri possesses seven putative pairs of σ/anti-σ factors belonging to the ECF family. Here, we report the identification of the genes regulated by five out of the seven A. butzleri ECF σ factors. Three of the ECF σ factors play an apparent role in transport, energy generation and the maintenance of redox balance. Several genes like the nap, sox and tct genes are regulated by more than one ECF σ factor, indicating that the A. butzleri ECF σ factors form a network of overlapping regulons. In contrast to other eubacteria, these A. butzleri ECF regulons appear to primarily regulate responses to changing environments in order to meet metabolic needs instead of an obvious role in stress adaptation.