Type IV Secretion: Adaptation of Conjugative Cellular Machinery

A number of pathogenic bacteria have adapted their conjugation machinery for protein secretion (16, 20). Bacterial conjugation machinery not only facilitates the transport of proteins to the extracellular matrix; in the case of H. pylori, effector molecules are injected directly into recipient eukaryotic cells (16, 20). This mechanism of bacterial conjugation machinery-mediated secretion is now widely known as type IV secretion. Most of our current knowledge of the conjugation apparatus is derived from the Agrobacterium tumefaciens T-DNA transfer machine, which delivers oncogenic nucleoprotein particles into plant cells (19, 43, 57, 58, 61, 78, 101, 102). Bacterial pathogens with known or putative type IV systems include Bordetella pertussis (20, 98), Legionella pneumophila (83, 85), Rickettsia prowazekii (4), and Brucella spp. (71).

The proteins comprising the type IV secretion systems are encoded by genes generally associated with pathogenicity islands, suggesting horizontal transfer of these conjugative systems (20). As discussed elsewhere in this volume, the genes associated with the H. pylori type IV apparatus are encoded within the cag (cytotoxin-associated gene) pathogenicity island (1, 17). The 145-kDa effector protein CagA is directly secreted from bacterial cells into epithelial cells via the cag-encoded type IV translocation apparatus. The strong correlation of the presence of the cag pathogenicity island with H. pylori strains associated with gastric disease, as well as the potential role of cag in bacterial pathogenesis, has been discussed in detail elsewhere in this volume. This chapter focuses on the cag genes linked to the H. pylori type IV secretion apparatus.

Components of the Type IV Secretion Apparatus

The conjugation machines of gram-negative bacteria are composed of two surface structures, the mating channel through which the cargo is transferred and the conjugative pilus for contacting recipient cells, along with membrane-associated ATPases (37, 98). High-resolution structural data are not available for any mating channel, although various conjugative pili have been visualized by lower resolution techniques such as electron microscopy (48). Most of the current information contributing to the model of bacterial conjugation machines derives from studies focused on interactions between specific protein subunits of the A. tumefaciens T-pilus transporter, as well as localization of individual transporter components in the assembled secretion machinery. The vir genes that encode the type IV secretion apparatus in A. tumefaciens can be divided into three functional groups that encode the following: (i) proteins comprising the mating channel, (ii) cytoplasmic membrane ATPases, and (iii) proteins that localize exocellularly to form the T-pilus or other adhesive structures (20, 58). Although these components are hypothesized to form a large supramolecular complex, there is not yet direct evidence for a physical association between the mating channel and the conjugative pilus.

Unraveling the structure of the H. pylori secretion apparatus represents a rich area for future research. Information about the functions and properties of specific Cag proteins have been derived primarily from sequence homologies with the Vir proteins of the A. tumefaciens conjugation machine (1, 17). It is striking that of the 31 genes identified within the H. pylori pathogenicity island, only six cag gene products demonstrate substantial amino acid similarities to the vir gene products (Fig. 1A). On the basis of biochemical and genetic studies of the A. tumefaciens secretion apparatus (58), the locations of the six H. pylori Cag proteins are predicted via analogy to their Vir homologs (Fig. 1B) to be either part of the mating channel, or, alternatively, one of the putative ATPases (Table 1). There are no Cag homologs to Vir proteins, which are involved exocellularly in contact or adhesion to host cells. The lack of additional homology between Vir and Cag proteins suggests that the H. pylori cag secretion apparatus may be substantially different from the type IV apparatus of A. tumefaciens. Alternatively, it cannot be ruled out that other Cag proteins may be able to carry out similar or identical functions to the Vir proteins in the A. tumefaciens secretion apparatus.

Figure 1

. H. pylori homologs of the A. tumefaciens conjugation machine. (A) Genetic organization of the A. tumefaciens conjugation machine. H. pylori type IV transporter components with homology to the Vir proteins are aligned underneath their homologs. (B) A (more...)

Table 1

H. pylori type IV system.

Homologs of A. tumefaciens mating channel components

The VirB6-VirB10 gene products from A. tumefaciens, as well as one or more of the three ATPases, are believed to be channel subunits (19). VirB7, an outer membrane lipoprotein (35), is homologous to H. pylori CagT; and VirB9 is a homolog to H. pylori Cag-528 (1, 17). VirB7 forms homodimers with neighboring VirB7 and interacts with VirB9 via reactive cysteines present in each protein that are oxidized to form disulfide linkages (3, 87), The VirB7-VirB9 heterodimer has been localized to the outer membrane and appears to be essential for stabilizing other VirB proteins during assembly of the A. tumefaciens transfer apparatus (35). By analogy to VirB7 and VirB9, CagT and Cag-528 may also be localized to the H. pylori outer membrane, and serve a role for assembly of the type IV secretion machinery in H. pylori.

H. pylori Cag-527 is homologous to A. tumefaciens VirB10 (Fig. 1A) (1, 17). VirB10 is believed to interact directly with VirB9 based on yeast two-hybrid analysis (24). In support of this hypothesis, VirB9 is essential for chemically cross-linking VirB10 into higher ordered multimers predicted to be homotrimers (6). It is thus reasonable to hypothesize that Cag-527 and Cag-528 (the VirB9 homolog) may interact within the H. pylori "mating channel" and carry out the same or similar functions during H. pylori type IV secretion as their homologs do in A. tumefaciens (Fig. 1B). Although VirB10 is proposed to bridge the cytoplasmic and outer membrane VirB subcomplexes (6), no evidence yet exists to support such a role for Cag-527.

Interestingly, there is no clearly identified H. pylori homolog to VirB6, which is a highly hydrophobic protein thought to span the cytoplasmic membrane several times (23). VirB6 is potentially the best candidate for a channel-forming protein in A. tumefaciens. Presumably, if the H. pylori type IV transfer apparatus is similar to that of the conjugation machine of A. tumefaciens, such a putative channel protein must also exist in H. pylori but may be more specifically tailored to the translocation requirements of this gastric bacterium.

Homologs of A. tumefaciens membrane ATPases

Type IV secretion requires functional cytoplasmic membrane ATPases. H. pylori CagE and Cag-525 are required for type IV translocation of effector molecules into mammalian cells and have considerable homology to the putative ATPase proteins of A. tumefaciens VirB4 and VirB11 (1, 17, 54). These VirB proteins contain conserved Walker A nucleotide-binding motifs required for function (19, 91). Purified forms of VirB4 and VirB11 possess weak in vitro ATPase activities (19). Both proteins appear to assemble minimally in vivo as homodimers. Dimerization of VirB4 is mediated by a domain in the amino-terminal third of the protein. In contrast, dimerization of VirB11 is mediated by domains located in each half of the protein (22, 77, 100). VirB11 and the H. pylori homolog Cag-525 have been shown to form higher ordered homohexameric rings in solution, further supporting the view that they may share similar or identical properties and functions (53, 77).

Another putative A. tumefaciens ATPase, VirD4, is believed to be important for DNA processing and transfer reactions by discriminating for transferred DNA (42). The H. pylori homolog of VirD4 is HP524, which is required for transport of the H. pylori protein effector molecule CagA into mammalian cells (89), suggesting that this family of putative ATPases may not be specific for the type of molecule transported by the type IV secretion machinery.

Homologs of A. tumefaciens exocellular T-pilus components

The third component of the A. tumefaciens secretion apparatus is composed of the exocellular VirB proteins localized to form the adhesive and contact structure for direct interactions with host cells (20, 58). There are no cag gene products that share significant sequence homology to A. tumefaciens proteins comprising the scaffold of the T pilus (1, 17). The lack of clearly identifiable homologs may represent differences in adaptive requirements for H. pylori and A. tumefaciens, which interact with gastric epithelial cells and plant cells, respectively. The proteins that comprise the exocellular structure required for contact and adhesion of the H. pylori type IV machine remain undefined, and their identification and characterization represent important areas for future investigations.

Bacterial Autolysis: An "Altruistic" Mechanism for H. pylori Adaptation within a Hostile Environment?

Nascent proteins targeted for secretion by type I or type II mechanisms are transported rapidly subsequent to their expression in the cytoplasm (70, 74). Consequently, proteins destined for secretion by these mechanisms are not generally localized simultaneously within the cytoplasm and extracellular milieu. In contrast, typical cytoplasmic H. pylori proteins, such as urease and catalase, have been found in the cytoplasm, localized to the outer membrane, and/or in the extracellular medium (14, 33, 73). This phenomenon has been observed in vitro, but it is thought that extracellular transport of H. pylori cytoplasmic proteins also occurs in vivo. Supporting this hypothesis are the observations that administration of urease, catalase, and other proteins confers protection against H. pylori challenge in animal models (11, 28, 30, 41, 59, 64, 68, 76).

Recent studies have suggested that specific H. pylori cytoplasmic proteins are localized to the outer membrane by a mechanism of bacterial autolysis (33, 73). According to this model (Fig. 2), individual bacteria are lysed by a highly regulated and controlled process that results in the release of cytoplasmic proteins from the bacterial cytosol. Notably, a number of other pathogenic bacteria, including Streptococcus pneumoniae and Neisseria gonorrhoeae, convincingly have been demonstrated to undergo a regulated program of autolysis (60, 69, 79, 84, 85). In H. pylori, some of the released cytoplasmic proteins are then adsorbed to the surface of the outer membrane of neighboring bacteria. Such a model has been called "altruistic autolysis" since the lysis of a fraction of the bacterial population presumably benefits the remaining viable bacteria (5, 32, 33, 73). This type of mechanism has been suggested by a number of investigators to explain how vaccination with a cytoplasmic protein such as urease can confer protection against challenges with Helicobacter spp. in animal models, as described (11, 30, 41, 59, 68). Because H. pylori is noninvasive, autolysis might also facilitate presentation of virulence factors and immunogens to the host (5, 32, 33, 73).

Figure 2

. H. pylori altruistic autolysis. This model predicts that H. pylori responds to specific environmental signals to activate genetically programmed bacterial autolysis. Specific protein autolysins degrade the H. pylori peptidoglycan layer to lyse the cell (more...)

Outer Membrane Vesicles: Bundling Cell Envelope Proteins for Delivery?

Recently, it has been shown that H. pylori releases small vesicles from its outer membrane by a process that bears striking similarity to the release of membrane vesicles by a number of other bacterial pathogens (36), namely, Neisseria meningitidis, H. influenzae, Borrelia burgdorferi, Pseudomonas aeruginosa, and Campylobacter jejuni (9, 15, 49–52, 62, 66, 99). H. pylori strains examined from gastric biopsies of infected patients generate small trilayered membranous vesicles, and it is hypothesized that they derive from the bacterial outer membrane (Fig. 3). Significantly, these outer membrane vesicles frequently contain numerous antigens and virulence factors. The production of these vesicles potentially represents an important mechanism for bacterial pathogens to modulate their environment within the host.

Figure 3

. H. pylori outer membrane vesicles budding. The release of outer membrane vesicles by H. pylori is an alternative mechanism for the delivery of bacterial toxins and antigens to the gastric mucosa. These small vesicles are 50 to 300 nm in diameter. They (more...)

Conclusions

Bacterial secretion remains a fertile area of research. H. pylori has classical type I and type II gram-negative bacterial secretion pathways, but it is becoming apparent that this bacterium uses alternative mechanisms for exporting proteins to the outside environment. The ability of a noninvasive bacterium such as H. pylori to modulate its environment is an important strategy for colonization and survival within the human stomach. Each of the mechanisms described above has the potential of allowing H. pylori to carry out important functions within the host. The type IV secretion system facilitates direct transport of protein effectors from the bacterium into the host cell. This adaptation of the conjugal machinery of many gram-negative bacteria bypasses the need for active uptake by the host cell. Bacterial autolysis probably involves mechanisms of peptidoglycan degradation to release cytoplasmic contents into the extracellular medium, which can then be strategically relocalized onto the outer membrane of other bacteria. In the case of H. pylori, localization of urease to the outer membrane appears to assist its survival in the acidic environment of the gastric mucosa. Finally, the release of outer membrane vesicles may allow H. pylori to bundle up cellular envelope proteins and deliver them to gastric epithelial cells and potentially to other locations distal to the site of colonization. Clearly, these three mechanisms represent remarkable adaptation strategies of H. pylori.

Acknowledgments

Research funding from the National Institutes of Health (ROI AI45928), the American Heart Association (98B 6472), and the Robert A. Welch Foundation (E-1311) is gratefully acknowledged.

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