High-frequency S-layer protein variation in Campylobacter fetus revealed by sapA mutagenesis

Campylobacter fetus utilizes paracrystalline surface (S-) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full-length S-layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full-length S-layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10^-2. Immunoblots of survivors showed that phenotypic reversion involving high-level production of full-length (98, 127 or 149 kDa) S-layer proteins had occurred. Revertants were serum resistant but caused approximately 10-fold less bacteraemia in orally challenged mice than did the wild-type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high-frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.     

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.     

Interbacterial macromolecular transfer by the Campylobacter fetus subsp. venerealis type IV secretion system

We report here the first demonstration of intra- and interspecies conjugative plasmid DNA transfer for Campylobacter fetus. Gene regions carried by a Campylobacter coli plasmid were identified that are sufficient for conjugative mobilization to Escherichia coli and C. fetus recipients. A broader functional range is predicted. Efficient DNA transfer involves the virB9 and virD4 genes of the type IV bacterial secretion system encoded by a pathogenicity island of C. fetus subsp. venerealis. Complementation of these phenotypes from expression constructions based on the promoter of the C. fetus surface antigen protein (sap) locus was temperature dependent, and a temperature regulation of the sap promoter was subsequently confirmed under laboratory conditions. Gene transfer was sensitive to surface or entry exclusion functions in potential recipient cells carrying IncPα plasmid RP4 implying functional relatedness to C. fetus proteins. The virB/virD4 locus is also known to be involved in bacterial invasion and killing of cultured human cells in vitro. Whether specifically secreted effector proteins contribute to host colonization and infection activities is currently unknown. Two putative effector proteins carrying an FIC domain conserved in a few bacterial type III and type IV secreted proteins of pathogens were analyzed for secretion by the C. fetus or heterologous conjugative systems. No evidence for interbacterial translocation of the Fic proteins was found.     

Complete genome sequence of type strain Campylobacter fetus subsp. venerealis NCTC 10354T

Campylobacter fetus subsp. venerealis is the etiologic agent of bovine genital campylobacteriosis, a sexually transmitted disease of cattle that is of worldwide importance. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. venerealis NCTC 10354(T) are reported.     

Genotyping and antibiotic resistance patterns of Campylobacter fetus subsp.venerealis from cattle farms in India

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is of considerable economic importance to the cattle industry worldwide. Cfv causes syndrome of temporary infertility in female cattle, early embryonic mortality, aberrant oestrus cycles, delayed conception, abortions and poor calving rates. In the present study, a total of 200 samples obtained from vaginal swabs, cervicovaginal mucous (CVM), preputial washes and semen straws were investigated that were obtained from organized cattle farm of MLRI, Manasbal and unorganized sectors. Out of a total of 200 samples, 49 (47·57%) vaginal swabs, 1 (3·33%) preputial wash and 8 (25%) carried out CVM samples were positive for Cfv, whereas none of the semen straws were positive for Cfv. A total of eleven isolates of Cfv were recovered. PFGE (Pulse field gel electrophoresis) analysis revealed four different pulsotypes (I–IV) circulating in the screened farms. A common pulsotype circulating among farms could not be established. Insertion element (ISCfe1), a 233 bp amplicon of Cfv, was sequenced and the sequence was deposited in GenBank (accession no: MK475662).     

A Genomic Island Defines Subspecies-Specific Virulence Features of the Host-Adapted Pathogen Campylobacter fetus subsp. venerealis

The pathogen Campylobacter fetus comprises two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis. Although these taxa are highly related on the genome level, they are adapted to distinct hosts and tissues. C. fetus subsp. fetus infects a diversity of hosts, including humans, and colonizes the gastrointestinal tract. In contrast, C. fetus subsp. venerealis is largely restricted to the bovine genital tract, causing epidemic abortion in these animals. In light of their close genetic relatedness, the specific niche preferences make the C. fetus subspecies an ideal model system to investigate the molecular basis of host adaptation. In this study, a subtractive-hybridization approach was applied to the genomes of the subspecies to identify different genes potentially underlying this specificity. The comparison revealed a genomic island uniquely present in C. fetus subsp. venerealis that harbors several genes indicative of horizontal transfer and that encodes the core components necessary for bacterial type IV secretion. Macromolecular transporters of this type deliver effector molecules to host cells, thereby contributing to virulence in various pathogens. Mutational inactivation of the putative secretion system confirmed its involvement in the pathogenicity of C. fetus subsp. venerealis.Campylobacter species are Gram-negative epsilonproteobacteria highly adapted to mucosal surfaces. The majority are human and/or animal pathogens (19, 61). The 18 species comprising the genus Campylobacter display a high degree of host and tissue specificity, which makes them excellent models to study host-pathogen relationships (25). The most prominent member, Campylobacter jejuni, is a commensal of the chicken intestine and the major cause of human bacterial diarrhea (74). Comparative analysis of Campylobacter genomes has revealed a process of genome decay—supported by a small genome size (about 1.5 Mb) and the loss of metabolic genes—consistent with successful adaptation to a specific niche (41). Campylobacter genomes are among the densest bacterial genomes known, with about 95% coding sequence. Despite this evidence of reduction, plasticity in genetic composition remains evident, as strain-specific genes comprise a substantial proportion of the entire repertoire of 1,500 to 1,800 genes (16, 23, 25, 56).This study focuses on the species Campylobacter fetus, which is represented by the two subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. Although the two taxa are genetically closely related, they exhibit striking tissue and host specificity. C. fetus subsp. fetus is a human, as well as animal, pathogen. Human infection results in serious systemic disease, especially in immunocompromised people. C. fetus subsp. fetus is the Campylobacter species most often isolated from human blood (75), and it is considered an emerging pathogen (9). The infection mode shares similarities with that of Salmonella enterica serovar Typhi. Orally acquired C. fetus subsp. fetus penetrates the intestinal mucosa, leading to bacteremia, and subsequent excretion via the biliary tract leads to secondary colonization of the intestine (9). Colonization of reproductive organs induces abortion in sheep and to a lesser extent in cattle, and very rarely in humans (11). C. fetus subsp. fetus can also be isolated from the intestinal tracts of birds and reptiles (78, 80). In contrast, C. fetus subsp. venerealis is host restricted. It is isolated primarily from the bovine genital tract and causes the epidemic disease bovine venereal campylobacteriosis (BVC). The reservoir of C. fetus subsp. venerealis is the penile prepuce of the bull. Transmission to cows occurs at coitus or during artificial insemination, and infection leads to endometritis, abortion, and infertility (28). Since BVC is a worldwide problem with substantial economic consequences, diagnosed cases must be registered (75) and import and export of bovine semen and embryos for cattle breeding requires statutory preclusion of C. fetus infection (2). Despite the distinct niche preferences of the C. fetus subspecies, they show high genetic relatedness, complicating the task of correct subspecies identification (46, 62, 81). Their population structure is clonal, and C. fetus subsp. venerealis is thought to represent a bovine clone of C. fetus (81).In this study, we employed the C. fetus subspecies to investigate the genetic basis for their host and tissue specificities. A genomic subtractive-hybridization approach was taken to identify subspecies-specific genomic fragments. This led to the discovery of a genomic island exclusively present on the chromosome of the host-adapted subspecies C. fetus subsp. venerealis. This island harbors a type IV secretion system (T4SS), as well as mobility genes (insertion sequence [IS] transposases and phage integrases) and shares substantial homology and similar structure with resistance plasmids found in other Campylobacter species. These features are indicative of a horizontally acquired genetic element. Finally, mutational analysis of genes within the island substantiates its involvement in C. fetus subsp. venerealis virulence.     

A deletion in the sapA homologue cluster is responsible for the loss of the S-layer in Campylobacter fetus strain TK

The surface array protein (SAP) of Campylobacter fetus strain TK is encoded by seven homologous sapA genes clustered on the chromosomal DNA. The spontaneously arising variant strain TK(SAP^–) produces no SAP and carries an approximately 10-kb chromosomal deletion. To elucidate the mechanism underlying the loss of SAP synthesis, we analyzed the region containing the sapA homologues and the deletion. We constructed a physical map of the sapA cluster region by aligning the clones that contain sapA homologues. These analyses demonstrated that all sapA homologues were located within a limited region of about 50 kb of chromosomal DNA of strain TK. The TK(SAP^–) deletion was located within this cluster and was 13.3 kb in size. The deletion occurred between two sapA homologues and resulted in the formation of a chimeric sapA homologue in the variant strain. Sequence analysis of the upstream regions and the conserved regions of all sapA homologues revealed a high degree of similarity. However, only one sapA homologue contained a putative promoter sequence. This promoter sequence was located in the deleted region. Thus, the deletion of the promoter appears to be responsible for the loss of SAP expression in TK(SAP^–). Received: 17 May 1996 / Accepted: 6 December 1996     

A new antibiotic combination for frozen bovine semen 1. Control of mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus

Systematic evaluations of new combinations of antibiotics for the control of bovine mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus in a bovine frozen semen process were made. These organisms were standardized to 10(5) to 10(6) colony forming unit (CFU) and inoculated into each ml of raw semen. Antibiotics in a final volume of 0.02 ml were added to each ml of the raw semen and were contained at the same concentration in the nonglycerol portion of the extenders (whole milk, 20% egg yolk citrate, 20% egg yolk tris, Plus-X, and 28% egg yolk tris). The combination of gentamicin (500 ug/ml) tylosin (100 ug/ml) and Linco-Spectin (300/600 ug/ml) was more effective for the control of mycoplasmas and ureaplasmas and equally effective for the control of C. fetus subsp. venerealis and Haemophilus somnus than the standard combination of penicillin, dihydrostreptomycin and polymyxin B sulfate.     

Contamination of red-meat carcasses by Campylobacter fetus subsp. jejuni.

Campylobacter fetus subsp. jejuni was commonly present in the feces of unweaned calves (2 to 3 weeks old) and from two of four groups of sheep. One new season lamb (12 to 16 weeks old) carried the organism, but the bacteria were not isolated from cattle. With unweaned calves, the fractions of animals infected and carcasses contaminated were similar. Contamination of carcasses usually involved low densities of C. fetus subsp. jejuni (ca. 1 to 10/cm2), which were isolated from flank but not rump areas. The organism was recovered less frequently from chilled carcasses and deboned veal. Small numbers of C. fetus subsp. jejuni could be recovered from equipment during the processing of unweaned calves but not after routine cleaning.     

Development of loop‐mediated isothermal amplification and PCR assays for rapid and simple detection of Campylobacter fetus subsp. venerealis

Campylobacter fetus is divided into CFV and CFF. Because CFV causes bovine genital campylobacteriosis, differentiation of the two subspecies is essential to the implementation of efficient CFV control and eradication programs. We have developed LAMP and duplex PCR assays for rapid and simple detection of CFV. The LAMP assay correctly detected 7 CFV strains and did not detect 53 CFF, 35 non‐fetus Campylobacter and 25 non‐Campylobacter strains. The PCR assay successfully differentiated the two subspecies. The LAMP and PCR assays were faster than conventional biochemical assays, requiring for detection less than 50 min and less than 4 hr, respectively, from the beginning of DNA extraction from a single colony on blood agar to final determination. Our LAMP and PCR assays are rapid and practical tools for detection of CFV.     

Differentiation of the subspecies of Campylobacter fetus by genomic sizing.

Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SalI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar "intermedius" and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.     

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.     

A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the conserved N terminus of a family of silent and divergent homologs.

Campylobacter fetus cells can produce multiple S-layer proteins ranging from 97 to 149 kDa, with a single form predominating in cultured cells. We have cloned, sequenced, and expressed in Escherichia coli a sapA homolog, sapA2, which encodes a full-length 1,109-amino-acid (112-kDa) S-layer protein. Comparison with the two previously cloned sapA homologs has demonstrated two regions of identity, approximately 70 bp before the open reading frame (ORF) and proceeding 550 bp into the ORF and immediately downstream of the ORF. The entire genome contains eight copies of each of these conserved regions. Southern analyses has demonstrated that sapA2 existed as a complete copy within the genome in all strains examined, although Northern (RNA) analysis has demonstrated that sapA2 was not expressed in the C. fetus strain from which it was cloned. Further Southern analyses revealed increasing sapA diversity as probes increasingly 3' within the ORF were used. Pulsed-field gel electrophoresis and then Southern blotting with the conserved N-terminal region of the sapA homologs as a probe showed that these genes were tightly clustered on the chromosome. Deletion mutagenesis revealed that the S-layer protein bound serospecifically to the C. fetus lipopolysaccharide via its conserved N-terminal region. These data indicated that the S-layer proteins shared functional activity in the conserved N terminus but diverged in a semiconservative manner for the remainder of the molecule. Variation in S-layer protein expression may involve rearrangement of complete gene copies from a single large locus containing multiple sapA homologs.     

Respiratory systems and cytochromes in Campylobacter fetus subsp. intestinalis.

Cell suspensions of Campylobacter fetus subsp. intestinalis grown microaerophilically in complex media consumed oxygen in the presence of formate, succinate, and DL-lactate, and membranes had the corresponding dehydrogenase activities. The cells and membranes also had ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity which was cyanide sensitive. The fumarate reductase activity in the membranes was inhibited by p-chloromercuriphenylsulfonate, and this enzyme was probably responsible for the succinate dehydrogenase activity. Cytochrome c was predominant in the membranes, and a major proportion of this pigment exhibited a carbon monoxide-binding spectrum. Approximately 60% of the total membrane cytochrome c, measured with dithionite as the reductant, was also reduced by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. A similar proportion of the membrane cytochrome c was reduced by succinate under anaerobic conditions, whereas formate reduced more than 90% of the total cytochrome under these conditions. 2-Heptyl-4-hydroxyquinoline-N-oxide inhibited reduction of cytochrome c with succinate, and the reduced spectrum of cytochrome b became evident. The inhibitor delayed reduction of cytochrome c with formate, but the final level of reduction was unaffected. We conclude that the respiratory chain includes low- and high-potential forms of cytochromes c and b; the carbon monoxide-binding form of cytochrome c might function as a terminal oxidase.     

Survival and growth of Campylobacter fetus subsp. jejuni on meat and in cooked foods.

Twelve strains of Campylobacter fetus subsp. jejuni isolated from humans and animals grew at temperatures ranging from 34 to 45 degrees C and pH minima between 5.7 and 5.9. Only one strain grew at pH 5.8 with lactic acid present at a concentration similar to that in meat. All strains had decimal reduction times of less than 1 min at 60 degrees C. Further examination of a typical strain showed that it grew at 37 degrees C on high-pH meat but not at 37 degrees C on normal-pH meat. Bacterial numbers on both high (6.4)-pH and normal (5.8)-pH inoculated meat declined at a similar rate when the meat was stored at 25 degrees C. At -1 degree C, the rate of die-off was somewhat slower on normal-pH meat but was very much slower on high-pH meat. The initial fall in bacterial numbers that occurred when meat was frozen was also greater for normal-pH meat than for high-pH meat. The organism exhibited a long lag phase (1 to 2 days) when grown in cooked-meat medium at 37 degrees C and died in meat pies stored at 37 or 43 degrees C. Evaluation of the risk of Campylobacter contamination of red-meat carcasses to human health must take into account the limited potential of the organism to grow or even survive on fresh meats and in warm prepared foods.     

Paired-end sequence mapping detects extensive genomic rearrangement and translocation during divergence of Francisella tularensis subsp. tularensis and Francisella tularensis subsp. holarctica populations

Comparative genome hybridization of the Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica populations have shown that genome content is highly conserved, with relatively few genes in the F. tularensis subsp. tularensis genome being absent in other F. tularensis subspecies. To determine if organization of the genome differs between global populations of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, we have used paired-end sequence mapping (PESM) to identify regions of the genome where synteny is broken. The PESM approach compares the physical distances between paired-end sequencing reads of a library of a wild-type reference F. tularensis subsp. holarctica strain to the predicted lengths between the reads based on map coordinates of two different F. tularensis genome sequences. A total of 17 different continuous regions were identified in the F. tularensis subsp. holarctica genome (CR(holar)(c)(tica)) which are noncontiguous in the F. tularensis subsp. tularensis genome. Six of the 17 different CR(holarctica) are positioned as adjacent pairs in the F. tularensis subsp. tularensis genome sequence but are translocated in F. tularensis subsp. holarctica, implying that their arrangements are ancestral in F. tularensis subsp. tularensis and derived in F. tularensis subsp. holarctica. PCR analysis of the CR(holarctica) in 88 additional F. tularensis subsp. tularensis and F. tularensis subsp. holarctica isolates showed that the arrangements of the CR(holarctica) are highly conserved, particularly in F. tularensis subsp. holarctica, consistent with the hypothesis that global populations of F. tularensis subsp. holarctica have recently experienced a periodic selection event or they have emerged from a recent clonal expansion. Two unique F. tularensis subsp. tularensis-like strains were also observed which likely are derived from evolutionary intermediates and may represent a new taxonomic unit.     

Aerobic and anaerobic respiratory systems in Campylobacter fetus subsp. jejuni grown in atmospheres containing hydrogen.

Maximum growth of Campylobacter fetus subsp. jejuni, strain C-61, occurred when the cultures were incubated with shaking in atmospheres containing approximately 30% hydrogen, 5% oxygen, and 10% CO2. Suspensions of cells grown under these conditions consumed oxygen with formate as the substrate in the presence of 0.33 mM cyanide, which completely inhibited respiration with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine and with lactate. Spectroscopic evidence with intact cells suggested that a form of cytochrome c, reducible with formate but not with lactate or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine, can be reoxidized by a cyanide-insensitive system. Analysis of membranes from the cells showed high- and low-potential forms of cytochrome c, cytochrome b, and various enzymes, including hydrogenase, formate dehydrogenase, and fumarate reductase. The predominant carbon monoxide-binding pigment appeared to be a form of cytochrome c, but the spectra also showed evidence of cytochrome o. The membrane cytochromes were reduced by hydrogen in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide at concentrations which prevented the reduction of cytochrome c with succinate as the electron donor. Reoxidation of the substrate-reduced cytochromes by oxygen was apparently mediated by cyanide-sensitive and cyanide-insensitive systems. The membranes also had hydrogen-fumarate oxidoreductase activity mediated by cytochrome b. We conclude that C. fetus jejuni has high- and low-potential forms of cytochrome which are associated with a complex terminal oxidase system.