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Chapter 23 : Pathogenesis of Campylobacter fetus
Category: Bacterial Pathogenesis
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Campylobacter fetus has been recognized as a significant pathogen of livestock for nearly a century. The genome sequence of C. fetus subsp. fetus strain 82-40 has recently been determined. Analysis of this sequence has confirmed and extended previous observations and has also provided new insights into C. fetus metabolism, physiology, and pathogenesis. The majority of the C. fetus N-linked general glycosylation pathway genes are also found in C. fetus, although unlike in C. jejuni, the genes are arranged in multiple clusters. The major pathogenesis-related difference of C. fetus compared with C. jejuni is the presence of the C. fetus S-layer. Surface array protein, type A (SapA) lacked an amino-terminal signal sequence that would direct its secretion to the cell surface. Before this, the only surface-layer protein (SLP) that lacked a signal sequence was that of C. crescentus. Observations made during the cloning of the sapA genes were important toward understanding the mechanisms by which the expression and antigenic variation of the encoded proteins are controlled. To investigate the role of the S-layer proteins in ovine abortion, an in vivo model was developed that used pregnant ewes subcutaneously challenged with C. fetus subsp. fetus strain 23D. The outcome of the infection in terms of effects on the fetus is dependent on the interaction between the pathogen and the host response. This model also provides a context to understand the role of S-layer proteins as virulence factors in human infections.
Model for C. fetus subsp. fetus disease of humans ( Blaser, 1998 ). C. fetus subsp. fetus is ingested from contaminated food, followed by colonization of the intestinal tract. Bacteremia can occur but in normal hosts is limited by the immune system. In compromised hosts, the bacteremia may be prolonged due in part to bacterial virulence factors such as its surface layer, which allows secondary infection of additional anatomical sites. These may subsequently serve as a source of bacteria for sustained or renewed sepsis. From Blaser (1998) .
Inhibition of complement factor C3 binding by C. fetus. 125I-labeled C3 was incubated with either C. fetus 23D (S+) or 23B (S–), and the amount of bound C3 determined. The S-layer in strain 23D prevents significant C3 binding. From Blaser et al. (1988) .
Electron microscopy of the C. fetus surface layer (S-layer). (A) Shown in ultrathin cross section ( Dubreuil et al., 1988 ), the S-layer appears as a ringlike structure external to the outer membrane (arrow). (B) In freeze-etch preparations of the cell surface ( Fujimoto et al., 1991 ), the S-layer appears as either regular tetragonal (left) or hexagonal (right) arrays. This micrograph demonstrates the ability of a single cell to express more than one type of S-layer. From (A) Journal of Bacteriology and (B) Infection and Immunity.
Structural features and comparison of DNA sequences of eight complete and one partial sapA homolog. The structure of each homolog is represented schematically by the aligned rectangles. Colored boxes identify shared regions of identity among the homologs. White boxes indicate nonconserved sequences, with no sequences >30 bp shared. The first 553 bp in all eight complete sapA homologs are shared. The Cf0007 ORF shared 752 bp with sapA7, but lacks the 5′ conserved region; it is referred to as sapAp8, as it is a partial homolog. From Molecular Microbiology.
Schematic representation of genes encoding type A and type B SLPs. The conserved 5′ regions of each gene are indicated by black (type A) or green (type B) rectangles. Colors show areas of conservation between homologs; white boxes represent homolog-specific sequences. From Tu et al. (2004a ).
DNA inversion events in a model system using a promoterless aphA (km) cassette inserted into the wild-type sapA2 locus (top line). When the sapA promoter is positioned in the proper orientation, resistance to kanamycin results in S– bacteria, at a frequency of 10–4 (second line). When kanamycin-resistant cells are removed from kanamycin selection and subjected to serum selection, S+ (serum resistant), kanamycin-sensitive cells arise at a frequency of 10–4 (third line). Solid arrows represent expressed genes, and broken arrows represent silent (unexpressed) genes. The stippled boxes represent the 600-bp conserved regions at the 5′ ends of sapA genes, and asterisks show the positions of the embedded inverted repeats that may play a role in the inversion process. The heavy line is the 6.2-kb invertible region. From Dworkin and Blaser (1996) .
Model for complex inversion events resulting in the expression of alternate sapA homologs. Simple inversion events ( Fig. 6 ) can occur, as well as more complex events in which the invertible region and one or more adjacent genes invert. Black boxes and other types of shading represent the conserved 5′ regions and divergent 3′ regions of sapA genes, respectively. The bent arrow shows the location of the unique sapA promoter, which is associated with the expression of the adjacent sapA homolog (straight arrow). The asterisks are sequences (χ, inverted repeats) that are potentially involved in the inversion process. From Dworkin and Blaser (1997b) .
Genetic organization of the 6.2-kb invertible region. Bold arrows indicate genes contained within the invertible region. Bent arrows represent the divergent sapA and sapCDEF promoters. Hatched lines denote the conserved 5′ regions of the flanking sapA homologs, indicated here as sapAx and sapAy. From Thompson et al. (1998) .
Schematic representation and genomic organization of the sap locus in C fetus strain 23D. The adjacent ORFs that are not sapA homologs (including sapC, D, E, F, and Cf0001...Cf0032) are indicated by shaded boxes. The PCR primers used in this study (PF, SF, TR, SR, AF-A7F, and AR-A7R) are designated by the arrows, which also denote the primer orientations. The horizontal line indicates the length of the fragment. From Molecular Microbiology.
Model for secretion and assembly of the S-layer. A hypothetical structure of the C. fetus SLP transporter is shown, based on similarities to other type I transporters. The putative stoichiometry of the SapE and SapF proteins in the assembled transport apparatus is based on data gathered for the E. coli HlyA transporter ( Thanabalu et al., 1998 ). Recognition of the SapA carboxy-terminal secretion signal is mediated by the SapD protein. The SapA/SapD complex initiates the sequential assembly of SapE and SapF trimers resulting in a contiguous pore through which SapA is secreted. SapA then may attach to LPS and be added to the growing S-layer.
Outcome of experimental ovine challenge with wild-type and mutant C. fetus strains
Biochemical properties of C. fetus S-layer proteins