A large number of putative lipoproteins have been found in the H. pylori genome (Tomb et al., 1997). Their abundance and potential for facilitating extensive linkages between inner and OMs has been hypothesized to explain the difficulty reported in experimentally separating inner and OM layers in most Helicobacter and Campylobacter species (O'Toole and Clyne, 2001). Few lipoproteins have been well-characterized in H. pylori, but those that have been studied play important roles in infection and the host immune response. One such lipoprotein is the highly conserved H. pylori adhesin A (HpaA), which was originally characterized as a neuraminyllactose-binding hemagglutinin (Evans et al., 1988, 1993), and was shown later to be a lipoprotein (O'Toole et al., 1995). HpaA has since been studied in depth, due to its conserved nature in most strains and its role in bacterial adhesion. Localization of HpaA has been highly disputed in the field, with some studies pointing to flagellar sheath localization (Luke and Penn, 1995), others showing cytoplasmic localization (O'Toole et al., 1995), and others simply showing membrane association (Bolin et al., 1995; Lundstrom et al., 2001). Due to its putative adhesin classification and putative membrane association, HpaA has been examined for use as a potential vaccine candidate (Flach et al., 2011). Similarly, another membrane-associated lipoprotein in H. pylori is Lpp20, first characterized as an 18-kilodalton OM antigen (Keenan et al., 2000), has been shown to be immunodominant in several studies (Keenan et al., 2002; Bakos et al., 2003). While these two lipoproteins are currently the only well-examined examples in H. pylori to date, the presence of several other recognized lipoproteins involved in the transport pathways of both LPS and OMPs (which will be discussed subsequently) adds additional importance to this class of membrane components.

The mechanism for lipoprotein trafficking has been well-characterized in E. coli (Matsuyama et al., 1995, 1997; Yakushi et al., 2000). In this system, lipoproteins are transported to the inner membrane via the Sec pathway, embedded into the membrane, and are subsequently sorted according to the presence or absence of an inner membrane (IM) retention signal (Masuda et al., 2002). Lipoproteins lacking the signal are transported to the OM. Early lipoprotein transport studies utilized spheroplasts that were generated by EDTA-lysozyme treatment, making them devoid of periplasmic proteins (Matsuyama et al., 1995). While these spheroplasts continued to secrete proteins from the cytoplasm that are normally destined for the periplasm and OM, the major OM lipoprotein (Lpp) was not secreted and remained in the IM, which suggested that a periplasmic factor was required for OM lipoprotein release from the IM (Matsuyama et al., 1995). When periplasmic-derived materials were added externally to the spheroplasts, Lpp was released from the IM, and purification of the periplasmic components identified the periplasmic chaperone LolA (Masuda et al., 2002). Further research involving the generation of various proteoliposomes went on to characterize the OM component of the trafficking pathway, LolB (Matsuyama et al., 1997), whose role is the insertion of lipoproteins into the OM. Further analysis identified integral membrane proteins LolC and LolE, as well as a soluble, cytoplasmic ATP-binding protein (LolD) that is recruited by the membrane-associated heterodimer LolCE to form a complex that acts as an ATP-binding cassette (ABC) transporter in the IM (Yakushi et al., 2000; Ruiz et al., 2006). Lipoproteins are taken up by this complex and their lipid moiety bound to LolA, which is believed to protect their fatty-acid chains from the aqueous environment of the periplasm. LolA then transports them to an OM-associated lipoprotein, LolB, which stabilizes their internalization into the OM. Exactly how LolB inserts lipoproteins into the OM has yet to be elucidated. LolA and LolB share similarity of structure, both possessing hydrophobic cavities believed to be the binding sites for the lipid moieties of lipoproteins (Takeda et al., 2003). The cavities are comprised of unclosed β-barrels with α helical lid domains thought to close when not in complex with any lipoprotein (Takeda et al., 2003). It is worth noting that LolC and LolE are highly similar in sequence, and that unlike the Lol complex found in E. coli, the integral membrane portion of the complex in N. meningitidis appears to consists of only a single LolCE homolog (encoded by NMB1235) instead of two. This can potentially be explained in that the single N. meningitidis protein homolog may simply form a homodimer to supplement for the missing protein component (Bos et al., 2007). Given the essential nature of most of these Lol complex components in E. coli (Narita et al., 2002) and the large number of lipoproteins encoded in the H. pylori genome previously mentioned (Alm and Trust, 1999; Baltrus et al., 2009), their use as potential antimicrobial targets is promising.

A single, IM component of the Lol complex from E. coli appears to be conserved in H. pylori (HP0787; Table 1), with highest similarity to LolE. As mentioned previously, LolE and LolC are highly similar in E. coli and it has been postulated that possessing only one of these proteins may be adequate for Lol complex formation (Bos et al., 2007), as in the N. meningitidis model. Insertion mutations in HP0787 exist (Salama et al., 2004), however this result indicating a potential lack of essentiality of this gene should be interpreted with caution, as the generation of deletion mutants for this gene have not been possible to date (Liechti and Goldberg, unpublished results). A few genome sequencing projects have assigned lolD designations to a putative H. pylori homolog (strain B8, HPB8_1387/HP0179) (Farnbacher et al., 2010), however, this characterization is based solely on the ABC transporter domain, and upon closer inspection, the N-terminus of the LolD protein (approximately 100 amino acids) is missing from this H. pylori gene (similar dubious gene designations have been assigned in C. jejuni). While no definitive LolD homolog appears to exist in H. pylori as such, ABC transport domains are notoriously divergent in this bacteria compared with other microbes (Tomb et al., 1997), and dozens of potential candidates with similar domain structure exist in the genome whose functions have not yet been identified (Tomb et al., 1997; Marais et al., 1999). In addition, LolB (the lipoprotein involved in inserting new lipoproteins into the OM in E. coli and N. meningitidis) appears to be absent in H. pylori, and in fact is missing in all ε proteobacteria sequenced to date. A protein with poor genetic similarity to LolA, the periplasmic chaperone component, does exist in H. pylori (HP0785, E value = 0.03) and has been shown to be non-essential (Chalker et al., 2001). Whether this protein acts similarly to the LolA characterized in E. coli has yet to be discerned, however amino acid similarity alignments between HP0785 and LolA show significant similarity for the core functional regions of the chaperone, with greatest similarity existing on one of the two sides of the open β-barrel domain (Figure 2). When examining the H. pylori amino acid sequence for the less well-conserved side of the β-barrel domain, amino acids that make up two of the β strands in this region in the E. coli structure are absent in the H. pylori sequence (Figure 2, shown in blue) potentially indicating a smaller, more tightly packed β sheet on that side of the open β-barrel domain.

The genetic orientation of the Lol complex-associated genes in ε proteobacteria is, in a word, perplexing. In E. coli, lolCDE (and in Neisseria, lolCD) are found in a single operon (Figure 3A), with lolB and lolA each existing separately, elsewhere in the genome (Yakushi et al., 2000). In most ε proteobacteria however, the single conserved homolog of lolC appears in an operon with secA, the cytoplasmic-localized membrane translocase. While this proximity to an essential gene may explain problems in targeting lolC for deletion in H. pylori, an added caveat exists. The H. pylorilolA homolog is, coincidentally, also located proximal to secA, albeit transcribed in the opposite direction (Figure 3B), potentially hinting at some selective element as of yet undetermined in either protein or lipoprotein trafficking in ε proteobacteria. Given the apparent absence of a lolD homolog at this locus combined with the presence of the membrane protein translocation ATPase (SecA) in its place, overlap in the Sec export pathway and the lipoprotein transport pathway in ε proteobacteria may be a distinct possibility.

It has previously been shown that a lipoprotein signal peptidase gene (HP0074) is essential in H. pylori, indicating that lipoproteins in general are important for H. pylori survival (Chalker et al., 2001). The presence of a potentially essential lolE homolog in the genome would support this hypothesis. The apparent absence of a lolB homolog in addition to lolA proving to not be essential however, indicates that if this transport pathway is still functional in H. pylori, that it may possess redundant/alternate protein components for lipoprotein periplasmic transport and OM insertion. Taken together, despite initial observations indicating a defunct pathway, the lipoprotein transport pathway in H. pylori is potentially viable, and given the prominence of lipoproteins encoded in the genome, this pathway represents a potential molecular target worthy of full characterization and further pursuit.

In most Gram-negative bacteria, the major target for the immune system during a bacterial infection is lipopolysaccharide (LPS) (Chow et al., 1999; Miyake, 2004). LPS is a key component of a Gram-negative OM, contributing to the structural integrity of the bacteria as well as providing a barrier against the host immune system. A number of cellular receptors recognize bacterial LPS and upon binding they induce an innate immune response (Caroff et al., 2002). The primary activator of the host response to the lipid A portion of LPS from E. coli is Toll-like receptor 4 (TLR4) (Chow et al., 1999). In many cases, the immune response resulting from the presence of LPS (also referred to as endotoxin) is so severe, that a dysregulated immune response to infection develops, known as sepsis, which can be life threatening. Studies are currently underway to develop molecular inhibitors of LPS, specifically targeting its ability to induce monocyte activation (Bosshart and Heinzelmann, 2007).

In examining the two predominant model microbes in the field of OM biogenesis, it is worth noting that differences in their LPS exist at a structural basis. While E. coli possesses true LPS, Neiserria expresses lipooligosaccharides (LOS) lacking the repeating O-antigen of most enteric bacteria; this distinction is thought to account for many of the dissimilarities in the immune response to the different bacteria (Zhang et al., 2007). H. pylori does produce a defined length O-chain attached to its core region whose length is variable between strains. Similar to LPS biogenesis in other bacteria, the O-chain of H. pylori is constructed in the cytoplasm and flipped to the periplasm where it is transferred to the core oligosaccharide by the O-antigen ligase (WaaL). Because the host's immune response to LPS is so robust, many pathogenic bacteria express an LPS structure that avoids detection. H. pylori is a well-studied example, as it can express the Lewis Blood Group (LBG) antigens and present them on the O side chain of its LPS (Appelmelk et al., 1996; Monteiro, 2000). LBG antigens are commonly associated with host monocytes, macrophages, granulocytes, and gastric epithelial cells. They are important for low-affinity adhesion to host gastric epithelial cells as well as in phagocytosis (Kirkland et al., 1997; Lepper et al., 2005) and it has been widely speculated that H. pylori uses this key feature to evade the host immune system (Appelmelk et al., 1996; Moran et al., 1999). The genes responsible for the creation of these bacterial LBG antigens are present throughout the genome, and many of them undergo phase variation (Wang et al., 2000). The variation generated in the bacterial population, whether due to phase variation or other mechanisms, results in the production of a wide array of differing LPS profiles within a given population. This is believed to be a “fine-tuning” of the bacterium's defenses, further enhancing the evasion of the immune response (Wang et al., 2000; Khamri et al., 2005; Salaun et al., 2005; Salaun and Saunders, 2006)

In addition, H. pylori modifies the lipid A portion of its LPS. When first characterized, the lipid A from H. pylori was found to be unique and distinguishable from that of enterobacteria in that there is an absence of a phosphoryl group at the 4′-position and fatty acyl groups at the 3- and 3′-position, as well as the presence of 2-aminoethyl phosphate at the 1-position (Suda et al., 1997). Further studies went on to show that H. pylori actually synthesizes two lipid A species, a minor one resembling that of E. coli (bis-phosphorylated and hexa-acylated) and a major species that contains fewer acyl chains, fewer phosphate groups, and a single 2-keto-3-deoxyoctonate (Kdo) sugar attached to the disaccharide backbone (Moran et al., 1997; Tran et al., 2005). This led to the discovery that H. pylori modifies its lipid A and that the result is a molecule with much lower immunobiological activity (Tran et al., 2005). Additional studies identified that while the lipid A from H. pylori is initially synthesized in this hexa-acylated form, a series of modifications after synthesis can result in a tetra-acylated molecule (Stead et al., 2005, 2008).

Lipid A is the “hydrophobic anchor” of the LPS, and is the biological initiator of septic shock (Raetz and Whitfield, 2002). Modifications of the lipid A portion of H. pylori LPS results in reduced immuno-reactivity by as much as 1000 times compared to the lipid A of other bacterial species (Ogawa et al., 1997, 2003a,b; Stead et al., 2005). The exact mechanism by which H. pylori LPS interacts with the immune system is currently in dispute. While it was initially characterized that the host receptor for H. pylori LPS was TLR4 (Kawahara et al., 2001) subsequent papers have shown that H. pylori interacts through TLR2 and not TLR4 (Smith et al., 2003). This controversial finding is apparently not limited to H. pylori, and the same phenomenon has been shown for other Helicobacters as well (Mandell et al., 2004). While another study exists, showing that chemically synthesized lipid A from H. pylori signaled via TLR4 (Ogawa et al., 2003a,b), highly purified H. pylori LPS in an alternate study showed activation only through the TLR2 complex (Yokota et al., 2007). TLR4 expression in gastric cell lines has also been used as evidence of H. pylori LPS activation (Kawahara et al., 2001; Su et al., 2003; Schmausser et al., 2004), however in a recent paper, it was shown that H. pylori LPS first signals via TLR2, which then acts through nuclear factor Y (NF-Y) to activate the transcription of TLR4 (Yokota et al., 2010). Whatever the mechanism, the presentation of LPS on the OM of H. pylori is essential for colonization and pathogenesis partially for to its role in membrane barrier function, but also for its unique ability to mask this pathogen from the host immune system.

The majority of the work on the LPS transport (LPT) pathway has been conducted in E. coli and N. meningitidis, and the pathways are summarized visually in Figure 1. Each model bacterial system provides differing tools that allow the system to be analyzed in a slightly different way. For example, LPS is not essential for N. meningitidis, allowing for comprehensive mutant studies to be conducted, while the E. coli system provides a high degree of technical genetic manipulation to artificially deplete components and analyze the effects. Both systems have shown that the transport pathway consists of two major membrane proteins, the flippase MsbA in the IM and the β-barrel IMP/LptD in the OM. MsbA allows for LPS in the cytoplasmic IM leaflet to be “flipped” onto the periplasmic leaflet. Once there, LPS is extracted from the IM in a process believed to be powered by an ABC transporter consisting of two integral membrane proteins (LptF and LptG) as well as an additional IM protein located solely on the cytoplasmic face of the membrane (LptB). LPS moves from the IM LptBCFG complex to the OM LptDE complex, traversing the periplasm via LptA (Ma et al., 2008; Tran et al., 2008). LptA recognizes the lipid moiety of LPS and is thought to protect the highly hydrophobic lipid element from the aqueous periplasm during transport. The energy source that powers this movement of LPS across the periplasm has yet to be elucidated.

From a genetic standpoint, the LPS transport pathway in H. pylori appears to be conserved in regard to the integral membrane proteins MsbA, LptB, and LptD (Table 1). The encoding genes (HP1082, HP0715, and HP1215/1216, respectively) are present in all sequenced H. pylori isolates available (strains 26695, J99, HPAG1) as well as Helicobacter acinonychis and Campylobacter jejuni and are highly conserved when compared with their E. coli homologs. Transposon mutagenesis studies have generated mutations in all three genes (Salama et al., 2004), however, whether the proteins are still functional in these mutants is not known.

Both msbA and lptD were found to be significantly up-regulated when H. pylori strain NTUH-C1 was treated with glutaraldehyde (Chiu et al., 2009) and ΔlptD and ΔmsbA mutants have been characterized in the same H. pylori strain (Chiu et al., 2007, 2009). These researchers determined that their mutants, derived from the clinical isolate NTUH-C1, were more susceptible to both n-hexane as well as several antibiotics (novobiocin, erythromycin, tetracycline, and penicillin) when compared to the parental strain, suggesting that MsbA and LptD play an important role in maintaining membrane integrity in H. pylori. Silver staining of proteinase-K digested whole cell lysates indicate lower amounts of total LPS present in ΔlptD, ΔmsbA, and ΔlptDΔmsbA mutants. Immunoblots using antibodies for Lewis antigens A and B on H. pylori LPS preparations were used to show the amounts of O-antigen in the preparations, and both antibodies showed significantly reduced amounts of O-antigen in all three mutants (Chiu et al., 2009). While these studies raise the potential that H. pylori can exist without LPS, as of yet, no examination of the lipid A of these mutants has been conducted. LptA and LptD from E. coli have been shown to interact with the lipid A moiety of LPS, indicating that it is lipid A that is actually the key component that is being transported by the Lpt transport pathway. There are examples in the literature of alterations in H. pylori lipid A resulting in a “truncation of the core oligosaccharide and loss of the attachment of the O sidechain containing Lewis X and Y” (Moran et al., 2004) and the expression of Lewis antigens in a cholesterol-dependent manner (Hildebrandt and McGee, 2009), however, no studies to date have shown that H. pylori can exist without lipid A in its OM, or have shown an alternate pathway for lipid A integration into the membrane. Strain specificity potentially plays a role with regard to the generation of membrane biogenesis mutants in H. pylori, as at present, generating directed insertion or deletion or mutants in lptD, msbA, or any of the other components of the Lpt complex in strains 26695, G27, 1061, or SS1 have proven unsuccessful (Liechti and Goldberg, unpublished results).

In searching for possible homologs for all the components of the LPS trafficking from E. coli in H. pylori, an interesting phenomenon found in all of H. pylori membrane trafficking pathways again presents itself. While the membrane spanning pathway components appear quite similar to their E. coli counterparts (Table 1), the components localizing to the periplasmic region of the pathway appear barely conserved and potentially non-existent in any recognizable form. Putative H. pylori homologs to LptF and LptG (HP0362 and HP1498, respectively) were only found after identifying homologs with either E. coli or N. meningitidis proteins in Helicobacter hepaticus (HH0602 and HH1496, respectively). The purported periplasmic LPS chaperone, LptA, found in both E. coli and N. meningitidis is present in H. pylori, albeit at a very low similarity, whereas the membrane-associated ABC transporter component LptB is highly conserved. Analysis of the putative H. pylori LptA shows high structural similarity at the central region of the protein, and interestingly the majority of divergence from the E. coli homolog exists at both the N- and C-termini (Figure 4). These regions of lower similarity comprise the putative points of interaction between LptA molecules when they form polymers, resulting in a twisted, β-jellyroll structure, in the presence of LPS (Suits et al., 2008). No obvious homologs exist for either LptC and LptE in H. pylori or any ε proteobacteria at the level of amino acid identity/similarity, however one report from the literature indicates that lptE is always present in a common locus in most bacterial genomes, and when it does not appear to exist on the level of genetic similarity, a putative lipoprotein can be found in its same genetic locale (Bos and Tommassen, 2011). In the case of H. pylori, a putative lipoprotein that shares common domain characteristics of LptE orthologs exists at this locus, directly downstream of leuS (Figure 5) and this appears to be the case for all other ε proteobacteria as well (Bos and Tommassen, 2011). When protein structural analysis software [RaptorX, (Peng and Xu, 2011)] is used to compare the putative H. pylori protein at the structural level to all solved crystal structures in the Protein Data Bank, the two most structurally similar proteins are characterized as “rare lipoprotein B” (early nomenclature for LptE) from Nitrosomonas europaea and Shewanella oneidensis (PBD designations 2JXP and 2R76), respectively. Similarly, a conserved protein in ε proteobacteria appears to exist in the same genetic locus as lptC (directly upstream of the lptA homolog) (Figure 6). Similarly, the H. pylori homolog to lptB (HP0715) is found directly upstream of rpoN, similar to its genetic localization in E. coli and N. meningitidis. Amino acid alignments show well-conserved residues in the N-terminus of the putative H. pylori LptC and poorer similarity past ∼ residue 110. The LptC periplasmic domain has been described as a “twisted boat” structure comprised of two β-sheets in opposition to each other (Tran et al., 2010) and the H. pylori putative protein possesses amino acid similarity through the first five β-strands (Figure 7). Unlike LptE, LptC is believed to be the crucial link between LptA and the ABC transporter (LptBFG) and as all localization and protein interaction data to date supports this hypothesis, the presence of a potential LptC-like protein in ε proteobacteria is encouraging.

H. pylori contains an OMP family consisting of approximately 33 genes. This family is divided into two subfamilies comprising of the H. pylori outer membrane protein (Hop) and the Hop-related (Hor) families (Alm and Trust, 1999). The vast majority of H. pylori OMPs belong to the Hop sub-family, including almost all known adhesion proteins that allow for the binding of H. pylori to numerous substrates from fucosylated histo-blood group antigens (Ilver et al., 1998) to host laminin (Senkovich et al., 2011). The protein family also contains a substantial number of porins (Exner et al., 1995; Doig et al., 1995; Peck et al., 2001) thought to play roles in nutrient uptake, though to date, no study has closely examined their function with regard to specificity of substrates. It is also worth noting that the flagella apparatus (Eaton et al., 1996) and chemotactic machinery (Foynes et al., 2000; Croxen et al., 2006) of H. pylori are both essential for colonization and both possess OM and IM protein components, as do the transport pathways for LPS and lipoproteins, not to mention the Cag and Com type IV secretion systems, required for injecting the CagA protein into host cells (Censini et al., 1996) and taking up DNA for homologous recombination (Stingl et al., 2010), respectively. It is quite extraordinary how almost everything required for the survival and pathogenesis of H. pylori (DNA and nutrient uptake, adherence, motility, chemotaxis, immune stimulation/evasion, VacA secretion, iron acquisition, etc.) requires β-barrel proteins in the OM; these proteins may all depend on a single mechanism by which they are inserted into the OM.

In attempting to use E. coli as a model to discern proteins involved in OM biogenesis, researchers needed to designate set criteria to judge whether a given protein was likely involved in the transport pathway. They reasoned that defects in membrane transport would result in a loss of barrier function and membrane permeability would significantly increase as a result (Braun and Silhavy, 2002; Ruiz et al., 2006). Therefore, alterations in membrane permeability became a key element to examine when considering potential candidate proteins for OMP transport. It was also shown early on that mis-folding of OMPs resulted in an envelope stress response, in which various chaperones involved in the protein transport pathway were up-regulated (Cosma et al., 1995; Raivio and Silhavy, 1997). Genes involved in the OM transport pathways were also assumed to exist in operons with other transport pathway genes in addition to being essential in E. coli and well-conserved in other bacteria. The ultimate test for proteins playing roles in OM biogenesis was depletion experimentation using either temperature-sensitive mutants or inducer-dependent expression systems. In both cases, levels of OMPs could be examined and locations within the cell detected and compared with wild-type bacteria. It was also shown that, in many cases, problems in OMP transport resulted in a down-regulation of membrane proteins, preventing the accumulation of mis-folded proteins at various stages of the transport process (Werner and Misra, 2005; Ruiz et al., 2006; Bos et al., 2007).

This basic understanding of membrane biogenesis became much less universal once similar studies were conducted in the bacterium N. meningitidis. In stark contrast to E. coli, N. meningitidis proved a more complicated model for recognizing new transport pathway components for a number of reasons. For example, N. meningitidis appears to completely lack the majority of proteins involved and essential for a periplasmic stress response (Bos et al., 2007), as characterized by the E. coli system. In addition, numerous genes identified as essential components of the OM biogenesis apparatus in E. coli are not essential in N. meningitidis (Bos et al., 2007). This does allow for the creation of mutants in this model system however, enabling a more straightforward, reductionist approach.

The sec pathway is a well-established system for trafficking proteins from the cytoplasm through the IM to the periplasm. In this transport pathway, chaperones SecA and SecB sequester proteins bound for the IM transport pathway by recognizing leader sequences on the designated protein (Kumamoto and Francetic, 1993; Valent et al., 1998). These chaperones then bring the newly formed protein to the membrane-localized portion of the Sec complex (SecDEFGY) (Van den Berg et al., 2004), which acts to move the protein through the IM. SecA then acts as the ATPase motor for this protein transport channel, providing energy for the mechanical “pushing” of the preproteins through the SecYEG complex (Tomkiewicz et al., 2006) and in so doing changes localization from cytoplasmic to peripheral IM and finally to integral IM. The leader sequence on the preprotein is removed by a leader peptidase, and the protein is then generally recognized by various periplasmic chaperones. In addition to the SecB-dependent transport of preproteins to the SecYEG complex, an alternate means of cytoplasmic transport exists (Koch et al., 1999) in the signal recognition particle (SRP) that recognizes either signal recognition peptides or the first transmembrane segment of proteins targeted for the IM. Once bound to the preprotein, the SRP complex migrates to the membrane, where a SRP receptor catalyzes the release of the preprotein and its insertion into the SecYEG complex. While initially it was thought that the majority of OMPs were SecB-dependent (Kumamoto and Beckwith, 1985), it has also been shown that OMPs misdirected to the SRP-dependent pathway can be secreted across the IM and are correctly assembled in the OM (Lee and Bernstein, 2001; Bowers et al., 2003).

Another IM transport pathway for proteins has also been characterized. Known as the Twin-Arginine Translocation (Tat) complex, this system involves three IM proteins (TatA, TatB, and TatC) that allow for protein transport independent of the Sec pathway (Lee et al., 2006). The general model for this complex is that TatA, with its multiple membrane spanning domains, forms a pore complex, while TatB and TatC stabilize the complex and allow for interactions with incoming cytoplasmic proteins. The three genes encoding these proteins are found in a single operon in E. coli and mutants have shown defects in IM trafficking for certain proteins (Lee et al., 2006). It has been postulated that the Tat pathway generally is utilized for proteins that undergo the majority of their conformational changes in the cytoplasm, as the transport pathway is relatively fast as compared with the Sec pathway. Studies have shown that mutations of genes encoding proteins involved in this pathway in various bacteria can result in a loss of infectivity resulting from the mislocalization of virulence determinants, which are normally moved through the periplasm by this mechanism (Voulhoux et al., 2001; Lee et al., 2006).

Recent E. coli studies have led to the discovery of an OM component of the OMP trafficking pathway, the Bam (YaeT) complex (Ruiz et al., 2006). Researchers, in an attempt to identify proteins potentially involved in membrane biogenesis, created an E. coli strain with a defective lptD allele. Barrier function of the OM in wild-type E. coli generally results in resistance to a number of antibiotics and various toxic compounds. However, E. coli strains containing mutations in key OM biogenesis genes, such as lptD, display a “leaky phenotype” in that their membranes are more permeable and as a consequence these mutants show increased susceptibility to toxins and antibiotics. By using the lptD mutant as a starter strain, and then selecting for spontaneous suppressor mutations that result in a return to wild-type levels of antibiotic resistance, researchers were able to characterize other proteins involved in the transport pathway (Ruiz et al., 2005). Among the mutations identified was the yfgL (bamB) gene, encoding an OM-associated lipoprotein. Co-immunoprecipitation studies revealed that BamB existed in a complex in the OM with three other lipoproteins; YfiO (BamD), NlpB (BamC), and SmpA (BamE), as well as the β-barrel protein BamA (Wu et al., 2005).

The homolog of BamA in N. meningitidis (Omp85) had previously been linked to OM biogenesis, and it was initially uncertain whether it also had an additional role in transport of LPS (Voulhoux et al., 2003; Voulhoux and Tommassen, 2004). Subsequent experimentation was able to show that LPS transport is affected when the overall levels of BamA are reduced. This effect was later found to be indirect, as the alterations in LPS transport were the result of the OMP of the LPS transport pathway (LptD) not being inserted correctly into the membrane (Bos et al., 2004). In both bacterial systems, bamA is an essential gene and is involved in the assembly of β-barrel proteins. In examining its genetic orientation, it was discovered that bamA is flanked by two other genes involved in membrane trafficking: the periplasmic chaperone skp as well as yaeL, a protease involved in the envelope stress response described earlier (Bos et al., 2007). Lipoprotein BamD was also shown to be essential for viability in E. coli and N. meningitidis, while mutations in the other three lipoproteins result in only mild alterations in membrane permeability (Onufryk et al., 2005; Wu et al., 2005). However, upon comparison of the pathway mechanics of both microbes, significant differences emerged. Depletion studies for BamA in E. coli showed no alteration in LPS trafficking, indicating no role for BamA involving LPS in E. coli (Doerrler and Raetz, 2005). It was also shown that depletion for BamA in N. meningitidis led to the accumulation of unfolded OMPs in the IM (Voulhoux et al., 2003) while in E. coli, depletion resulted in significantly reduced amounts of all OMPs (Wu et al., 2005). It is believed this is due to the absence of the envelope stress response in N. meningitidis, thereby preventing the down-regulation of OMP synthesis (Bos et al., 2007). Subsequent studies have also shown that BamA also acts as the receptor for contact-dependent growth inhibition (CDI) in E. coli, and that antibodies specific for extracellular epitopes on BamA block inhibitor cell binding (Aoki et al., 2008). The potential for utilization of CDI to target bacterial growth is currently being examined (Aoki et al., 2011).

The four major periplasmic proteins involved in OMP transport in E. coli are SurA (Rouviere and Gross, 1996), DegP (Misra et al., 1991; Krojer et al., 2002), FkpA (Dartigalongue et al., 2001), and Skp (Harms et al., 2001; Bulieris et al., 2003; Walton and Sousa, 2004). Unlike various other members of the OM biogenesis pathway, Skp as well as other periplasmic chaperones, have been shown to be non-singularly essential, and mutants in E. coli show only modest defects in membrane biogenesis (Rizzitello et al., 2001). This has led to the belief that potentially multiple mechanisms exist for periplasmic trafficking, a view supported by greater OM defects seen in sequential knockout studies for other established periplasmic chaperones (Rizzitello et al., 2001), indicating the presence of parallel pathways. To date, SurA is recognized as the main periplasmic chaperone, with Skp/DegP acting as a back-up pathway (Sklar et al., 2007). Evidence does however exist in the literature that some proteins appear to be preferentially transported by SurA (Vertommen et al., 2009). LptD is currently believed to be transported through the periplasm by the SurA chaperone with cooperation from periplasmic disulfide isomerase (DsbC) and thiol oxidase (DsbA) (Denoncin et al., 2010). Further analysis revealed that LptD contains two disulfide bonds that are essential for function that act to stabilize the LptD protein by correctly positioning the N- and C-terminal domains of the protein in the periplasm (Ruiz et al., 2010) and that oxidation is carried out by DsbA. However, this apparent preference for periplasmic chaperones is potentially species-specific, as ΔsurA mutants generated in N. meningitidis did not appear to affect membrane biogenesis, while a Δskp mutant resulted in significantly lower levels of porins PorA and PorB (Volokhina et al., 2011).

A large number of the membrane-associated proteins found on the surface of H. pylori consist of β-barrels, indicating their transport through the membranes from the cytoplasm may be through the Sec and BamA complex (Table 1). The importance of the Sec pathway in H. pylori was made readily apparent via bioinformatic analysis of the H. pylori strain 26695, where it was found that 517 of the 1,590 open reading frames possessed putative signal sequences (Alm et al., 1999). The Sec pathway for IM transport has already been well-characterized in H. pylori and transposon mutagenesis studies have shown that many of the genes encoding components of the Sec-pathway are capable of being mutated (albeit definite conclusions based on these results should be tempered with the knowledge that often insertions in a gene do not always lead to a complete inactivation of that gene), with the exception of secY (Salama et al., 2004). H. pylori does seem to lack the cytoplasmic chaperone SecB, but this is apparently common and has been characterized in other bacterial species (Yamane et al., 2004), indicating SecB acts to supplement the effectiveness of SecA. This may also hint at a more pronounced role for the SRP-dependent pathway in OMP trafficking to the Sec complex in H. pylori, however, to date no studies have tested this potential hypothesis. E. colisecD and secG temperature-sensitive mutants when grown at the non-permissive temperature result in the accumulation of OMPs in the cytoplasm (Fitchen et al., 2003). However, when complementation studies were preformed using plasmids expressing the H. pylori homologues in these mutants, cytoplasmic OMP levels returned to their wild-type levels, indicating a conservation in function for these genes in H. pylori (Fitchen et al., 2003).

Similarly, the Sec-independent IM transport system, the Tat complex, appears to be well-conserved in H. pylori. tatA (HP0320), tatB (HP1060), and tatC (HP1061) are all present (Table 1), with the only difference being the apparent removal of tatA from the original operon and insertion elsewhere in the genome, whereas in E. coli the three genes comprise a single operon (Lee et al., 2006). No characterized mutants for tatB or tatC have been described while a single transposon insertion mutant exists for tatA (Salama et al., 2004) and the low coverage of transposon mutants in these genes suggests that all three maintain an essential function in H. pylori. This would seem to differ with previous studies in E. coli where the Tat system has been shown to be dispensable under laboratory conditions, however, there are examples of bacteria that rely heavily on the Tat system for almost all protein secretion (Dilks et al., 2005).

The periplasmic chaperones DegP and SurA appear to be conserved in H. pylori, while the chaperone Skp does not appear to be well-conserved. N. meningitidis is similar to H. pylori in this respect, as it posseses only surA and skp homologs and is missing a functional degP homolog (Volokhina et al., 2011). The synthetic lethal phenotypes characterized in E. coli double mutants (surA/skp and surA/degP) indicate that SurA and Skp/DegP exist in parallel periplasmic chaperone pathways, and the absence of a Skp homolog in H. pylori is possible due to the presence of SurA. No mutants in degP or surA have been characterized to date in H. pylori however, and insertional mutants in these genes were not generated in the construction of a transposon library in H. pylori of ∼10,000 clones (Salama et al., 2004), indicating that they may be essential.

The integral OMP component of the OMP trafficking pathway, BamA, is well-conserved in H. pylori (Table 1) and all ε proteobacteria. What is striking is that in E. coli and N. meningitidis, this protein is part of a complex along with at least four other conserved lipoproteins (BamB, BamC, BamD, and BamE) while in H. pylori only BamD (ComL) appears to be present. It is worth noting that BamA and BamD appear to form the core of the OMP export machinery, as they are the only two essential components in both E. coli and N. meningitidis. In E. coli BamE has been recently shown to modulate the conformation of BamA, likely through its interactions with BamD, and when Δ bamE mutant cells are treated with proteinase K, BamA is degraded beyond recognition (Rigel et al., 2012). No studies on the Bam complex have been reported in H. pylori to date.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.