Plasticity of repetitive DNA sequences within a bacterial (Type IV) secretion system component

Abstract

DNA rearrangement permits bacteria to regulate gene content and expression. In Helicobacter pylori, cagY, which contains an extraordinary number of direct DNA repeats, encodes a surface-exposed subunit of a (type IV) bacterial secretory system. Examining potential DNA rearrangements involving the cagY repeats indicated that recombination events invariably yield in-frame open reading frames, producing alternatively expressed genes. In individual hosts, H. pylori cell populations include strains that produce CagY proteins that differ in size, due to the predicted in-frame deletions or duplications, and elicit minimal or no host antibody recognition. Using repetitive DNA, H. pylori rearrangements in a host-exposed subunit of a conserved bacterial secretion system may permit a novel form of antigenic evasion.

Figure 1.

Direct nucleotide repeats in the structure of H. pylori cagY. (A) The abscissa represents the nucleotide position in each ORF and the ordinate for each of three strains (26695, J99, NCTC11168) represents the number of times each nucleotide is part of a direct repeat sequence ≥16 nucleotides. For each strain, the repeats are clustered in two regions. (B) The abscissa represents the nucleotide position in each ORF and the ordinate represents the number of times that identical repeats ≥16 bp flank each nucleotide. The presence of a nucleotide between identical repeats indicates that it could be deleted or duplicated if recombination occurred between the repeats. (C) For the three strains analyzed, cagY might be separated into five defined regions present in each sequence, which we termed the FRR, the FCR, the MRR, the TCR, and the VHR. None of the repeats are shared in both the FRR and MRR. Downstream of both the FRR and MRR are highly conserved regions of ∼550 bp (FCR and TCR), followed by the VHR portion of cagY, encoding a polypeptide highly homologous to the A. tumefaciens virB10 product. The FCR, TCR, and VHR have high levels of DNA identity between strains, with FCR size differences due to small variations in the boundaries of the adjacent repeat regions. FRR size variation is largely due to a 329-bp deletion (hatched region). The G+C content is lowest and deviates substantially from the remainder of the genome in the portion of cagY upstream of the MRR.

Figure 2.

Size and sequence diversity in the cagY MRR of unrelated H. pylori isolates. PCR was performed using 26695 primers FRRF and FRRR that flank the FRR, or using primers MRRF and MRRR that flank the MRR, and products resolved on a 1% agarose gel. The variation in these 12 strains is representative of studies of a global collection of 62 unrelated H. pylori strains.

Figure 3.

In vitro (A) and in vivo (B) cagY allelic diversity among H. pylori isolates having the same clonal origin. (A) PCR using primers MRRF and MRRR performed on 142 84–183HP (high passage) single colony isolates identified three cagY allele populations with products of 2.5 kb (lane 2), 2.0 kb (lane 3), and 2.0, 2.2, and 2.5 kb (lane 4) compared with identification of a single cagY allele population (unpublished data) within strain 84–183LP. Immunoblots of the three representative alleles using α-CagY serum show the expected protein size variation (lanes 2 and 4) and the mixed alleles (lane 4). PCR and immunoblots performed on a control cagY ⁻ strain, 88–22l, yielded no product (lane 1). (B) RFLP patterns in the cagY MRR in 14 H. pylori isolates from 7 members (patients 2–8) of an extended family from The Netherlands (reference 31). H. pylori glmM (control) and the cagY MRR were PCR amplified from the isolates and the products were digested with MboI for 3 h and electrophoresed on a 1% agarose gel.

Figure 4.

Size and sequence diversity in the cagY MRR in paired H. pylori isolates obtained 7.4 yr apart from an individual host. (A) The cagY MRR was amplified from isolates 13aqs (original strain) and 13bqs (obtained 7.4 yr later). (B) Sequence analysis of the PCR products showed that relative to strain 13aqs, strain 13bqs has a 324-bp deletion including at its 5′ end a 156-bp sequence (underlined) identical to the 156-bp sequence immediately 3′ of the deletion. Both copies of the repeat are present in 13aqs. (C) Translation of 13aqs and 13bqs indicate that cagY is in-frame in both strains.

Figure 5.

In vivo cagY MRR deletion occurs during experimental colonization of FVB/N mice. (A) CagY MRR PCR performed on single colonies from H. pylori strains before and after 8 mo of challenge of FVB/N mice. Lane 1, PCR⁻ control; lane 2, B128 prechallenge strain (sweep of colonies); lanes 3–7, single colonies from B128 prechallenge strain; lane 8, cells recovered from mouse 6.11 (sweep of colonies); lanes 9–13, single colonies from mouse 6.11; lane 14, cells recovered from mouse 6.16 (sweep of colonies); lanes 15–18, single colonies from mouse 6.16. (B) Sequence analysis of the PCR products showed that relative to strain B128, strain 6.16 has a 441-bp (in-frame) deletion, including at its 3′ end a 33-bp sequence (underlined) identical to the 33-bp sequence immediately 5′ of the deletion. (C) Immunoblotting with α-CagY rabbit serum of cell lysates from H. pylori strains before and during the experimental challenge. Lane 1, B128 strain before challenge; lane 2, B128 isolated from mouse 6.11; lane 3, B128 isolated from mouse 6.16.

Figure 6.

CagY protein size varies in H. pylori strains. Whole cell lysates from nine wild-type H. pylori strains were subject to immunoblot using α-CagY serum. For each cag ⁺ strain, multiple CagY products ranging from ∼180–220 kD were identified. In these strains, the CagY bands differ in size, number, and intensity. Strain 26695ΔrecA yielded multiple CagY bands, indicating that variation does not require RecA-dependent cagY deletion or duplication. The complete absence of bands in cag ⁻ strains 88–22 and in 26695Δcag confirms the specificity of the antibodies.

Figure 7.

Serum IgG responses to H. pylori CagA and CagY antigens. (A) By immunoblot, the recombinant J99 CagY antigen was recognized by rabbit α-CagY serum (lane 1), but not by serum from the patient from whom strain J99 was isolated (lane 2). (B) Similarly, using whole cell lysates from strain J99, the rabbit α-CagY serum (lane 1) recognized multiple high molecular weight CagY bands, whereas patient J99 (lane 2) recognized multiple bands (including CagA, UreB, and UreA), but not CagY. (C) That α-CagY immune rabbit sera reacted with the CagY antigen whereas control rabbit sera did not, indicates the antigenicity of CagY in the ELISA format. To determine whether the CagY protein is recognized by immune responses in persons with persistent H. pylori colonization, sera were examined from 38 H. pylori ⁺ persons for IgG antibodies to CagY and to CagA (as a control). As expected, sera from 27 patients carrying cagA ⁺ strains reacted more strongly (P < 0.001) to a recombinant CagA antigen than did sera from 11 patients carrying cagA ⁻ strains. Comparison of IgG responses to the CagY antigen between persons carrying cagA ⁺ and cagA ⁻ strains showed heterogeneous, but generally low level, responses. Although several patients had limited recognition of the recombinant CagY protein, the two groups of patients were not significantly different (P = 0.11).