Exploring the Role of Integral Membrane Proteins in ATP-Binding Cassette Transporters: Analysis of a Collection of MalG Insertion Mutants

The maltose transport complex of Escherichia coli is a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK₂ assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK₂ complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.Integral membrane proteins play a central role in the ATP-binding cassette (ABC) transporter superfamily, whose prokaryotic and eukaryotic members traffic a variety of substrates such as ions, sugars, amino acids, peptides, and proteins (15). This large family of transporters is defined by a conserved cytoplasmic ATPase component and integral membrane domains which interact to carry out the specific transport process (4, 15). Among the eukaryotic members are such medically relevant proteins as the P-glycoprotein implicated in multidrug-resistant cancer cells, the cystic fibrosis transmembrane regulator protein, and the human peroxisomal adrenoleukodystrophy protein (2, 34, 35). Among the prokaryotic members of the ABC superfamily are the periplasmic binding protein-dependent transporters. These family members are characterized by a conserved region of the integral membrane component(s) in addition to the conserved cytoplasmic ATPase (4). One member of this prokaryotic subgroup, the maltose transport complex of Escherichia coli, presents a useful model for the integral membrane folding and assembly interactions required for ABC transporters. The maltose transport complex consists of the integral membrane proteins MalF and MalG and a peripheral cytoplasmic membrane ATPase, MalK (reviewed in reference 24). These three proteins copurify (11), forming a MalFGK₂ tetrameric complex which acts in concert with the periplasmic maltose-binding protein (MBP), the product of malE, to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane.MalF has been shown to have eight transmembrane (TM) domains (5), whereas MalG possesses six TM domains (6, 10). Following independent insertion of these proteins into the membrane (22a, 31), assembly of the MalFGK₂ complex is likely mediated by interactions among discrete domains of MalF, MalG, and MalK, resulting in tetramerization (20, 26).Although the specifics of these interactions are unknown, a combination of biochemistry and genetics has allowed for a partial characterization of the complex. Shuman and colleagues isolated and characterized MalF and MalG mutants which enable the MalFGK₂ complex to transport maltose in the absence of MBP (7, 32). These analyses have pointed toward a direct interaction between MBP and periplasmic portions of MalG and MalF (16), between MalG and MalF themselves (7), and between MalK and both MalF and MalG (12). Davidson and Nikaido purified the MalFGK₂ complex and demonstrated extensive chemical cross-linking between MalG and MalF and among MalG, MalF, and MalK (11). Traxler and Beckwith observed that periplasmic loops of MalF become protease resistant only in the presence of MalG and MalK, also suggesting that specific interactions occur among the proteins in the context of an assembled complex (31). Finally, a potentially important MalG-MalK protein interaction signal has been identified in the hydrophilic cytoplasmic loop between the fourth and fifth TM domains of MalG (reference 9; Fig. ​Fig.1).1). This motif is conserved in MalF and in other binding protein-dependent transporters of the ABC superfamily (9, 28) and has been hypothesized to mediate interactions with the conserved ATPase subunit of the complex (17, 22). Open in a separate windowFIG. 1Topology model of MalG. Hydropathy plots and fusion protein analyses (6, 10) suggest that the N and C termini of the 296-residue protein are cytoplasmically localized. The shaded boxes represent putative TM domains, and the shaded amino acids are conserved in integral membrane proteins of periplasmic binding protein-dependent ABC transporters (9, 28). The location of each 31-residue insertion is shown by an arrowhead. The black arrowhead represents an insertion which did not significantly affect MalG transport function, the gray arrowhead depicts partial transport function, and the white arrowheads represent loss of transport ability for the corresponding insertion mutants. Each numbered disc shows the mutant classification of the adjacent insertion mutant (see Discussion for details).Recently, a transposon-mediated insertion mutagenesis technique was developed and used to characterize both permissive and nonpermissive regions of the integral membrane protein LacY (19), as well as the cytoplasmic MalK and LacI proteins (18, 23). These analyses not only identified tolerant hydrophilic regions of each protein but also defined several distinct mutant classes (18, 19, 23). In particular, the phenotypes attributable to the lacI insertion mutations that we isolated were strikingly similar to those of previously characterized amino acid substitutions mapping to the same sites in lacI. Here, we describe the results of this insertion mutagenesis on the MalG protein. This analysis provides a unique in vivo view of the requirements for proper MalG protein folding and of the interactions necessary for MalFGK₂ assembly and maltose transport.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Proteomics Identification of Drosophila Small Interfering RNA-associated Factors

The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an ∼80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.Small RNAs can regulate gene expression through a collection of mechanisms broadly termed RNA silencing. Small RNA-mediated silencing mechanisms occur in most species (1–5). The ability to silence the expression of specific genes using small RNAs via RNA interference (RNAi)¹ has greatly facilitated our understanding of gene function in eukaryotes. In addition, small RNA-mediated gene silencing has therapeutic potential and holds promise for the treatment of specific diseases (6). Understanding the mechanism of RNAi and identifying the components of the RNAi machinery are essential for harnessing its full potential in both genome-wide screens and therapeutic applications.Recently, high throughput sequencing technology has revealed the presence of endogenous siRNAs in plant, fly, worm, and mammalian cells (7–16). These endogenous siRNAs target transposable element RNAs, pseudogene RNAs, and protein-coding mRNAs (17). Therefore, the endogenous siRNA pathway seems to have evolved as a mechanism of cellular defense against selfish genetic elements. The roles of these siRNAs in development and cell physiology are poorly understood.Drosophila melanogaster is a well characterized model system for studying RNAi. In Drosophila, long double-stranded RNAs (dsRNAs) are processed by the endonuclease Dicer-2 into 21-nucleotide siRNAs (18). After processing, these siRNAs form an initiator complex with Dicer-2 and the dsRNA-binding domain (dsRBD)-containing protein R2D2 (19–23). This R2D2-Dicer-2 Initiator (RDI) complex transitions to a larger siRNP called the RISC loading complex (21, 22, 24, 25) and then to pre-RISC (26). Subsequently, pre-RISC matures into holo-RISC, which includes the catalytic activity necessary for target mRNA cleavage (21, 25, 27). The endonuclease subunit responsible for target cleavage in holo-RISC is Argonaute-2 (Ago2) (28, 29), which uses the guide strand of the siRNA duplex to target complementary mRNA sequences for cleavage and degradation.Studies of the RDI complex strongly suggest that it includes no other proteins besides Dicer-2 and R2D2 (22). Additional proteins such as Ago2 are present in pre-RISC and holo-RISC, but nonetheless the complete compositions of the RISC loading complex, pre-RISC, and holo-RISC are unknown. Furthermore, holo-RISC sediments at ∼80 S during sucrose gradient centrifugation (30). These observations indicate that additional protein factors associate with siRNAs. In this study, we identified siRNA-binding proteins from Drosophila embryo extracts. Target cleavage assays and immunoblotting of our siRNA affinity-selected proteins suggest that we purified active holo-RISC components. Proteomics analysis of the affinity matrix revealed both established and novel siRNA-associated proteins. Functional analyses of a subset of these factors suggest that they play important roles in RNAi.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species.Gram-negative bacteria have evolved several specialized protein secretion systems to secrete a wide variety of substrate proteins into the extracellular milieu or to inject them into other, often eukaryotic, cells (1). Secreted proteins and their associated secretion systems are very important in bacterial virulence and interactions with other organisms (2). One of the most recent discoveries in this field is the Type VI secretion system (T6SS),¹ which occurs widely across bacterial species (3, 4) and can target proteins to both bacterial and eukaryotic cells (5). The significance of the T6SS is becoming increasingly apparent. It has been implicated in virulence, commensalism, and symbiosis with eukaryotes (5, 6). Additionally, in many bacteria, the T6SS is now implicated in antibacterial activity. T6SS-mediated antibacterial killing appears to be important for competition between bacterial species, for example within the resident microflora of a eukaryotic host (5, 7).Secretion by the T6SS relies on 13 conserved core components which are predicted to form a large machinery associated with the cell envelope, including membrane-bound and bacteriophage tail-like subassemblies (8, 9). The membrane bound subassembly consists of inner membrane proteins (TssLM) and an outer membrane lipoprotein (TssJ) and is anchored to the cell wall. The phage tail-like assembly consists of several proteins that show structural homology with T4 phage tail proteins or are organized in similar structures (10). Hcp (TssD) proteins form hexameric rings and are thought to stack into tube-like structures (11, 12). This Hcp tube is believed to be capped by a trimer of VgrG (TssI) proteins, which share structural homology with the needle of the T4 phage tail (10, 13). In addition, VipA (TssB) and VipB (TssC) form a large tubular structure highly reminiscent of the T4 phage tail sheath (14, 15). Such similarities have led to the idea that the T6SS resembles an inverted contractile bacteriophage infection machinery and injects substrates via an Hcp/VgrG needle into other cells. Recent models propose that the VipA/B sheath surrounds the Hcp/VgrG needle and contraction of the VipA/B tube pushes the Hcp/VgrG needle out of the cell (16–18). It has been postulated that this mechanism can be triggered by close contact with other neighboring cells (19–21).Assembly, localization, and remodelling of VipA/B tubules in vivo depend on the AAA+ ATPase ClpV (TssH), another essential core component of the T6SS (14, 16, 17). ClpV also interacts with the accessory component Fha (TagH) (22, 23), which is found in a subset of T6SSs (4). The Fha protein has an N-terminal domain with a forkhead associated motif, which is predicted to bind phospho-threonine peptides (24). In Pseudomonas aeruginosa, Fha1 is phosphorylated by the Thr/Ser kinase PpkA (TagE) and dephosphorylated by the phosphatase PppA (TagG), and the phosphorylation state of Fha1 regulates the activity of the T6SS (22, 23). Phosphorylation of Fha in P. aeruginosa is also controlled by additional components, which act upstream of PpkA and form a regulatory cascade for T6SS activation (22, 25). Although homologs of PpkA and PppA have been identified in the T6SS gene clusters of certain other bacteria (3), the regulation of the T6SS by post-translational protein phosphorylation has not yet been experimentally investigated outside of Pseudomonas.To understand how the T6SS affects eukaryotic and bacterial cells, it is critical to identify substrate proteins secreted by the T6SS. The VgrG and Hcp proteins were the first identified T6SS substrates and appear to be generally secreted to the external milieu by all T6SSs (26). However, as mentioned above, Hcp and VgrG are core components of the T6SS machinery and therefore represent extracellular components of the secretion apparatus rather than genuine secreted effector proteins. Nonetheless, a limited number of VgrG homologs with extra functional effector domains at the C terminus have been identified or predicted, which account for some of the T6SS dependent effects seen against bacteria and eukaryotes. For example, the C-terminal domain of VgrG-1 from Vibrio cholerae shows actin crosslinking activity in eukaryotic cells (13, 27) and the C-terminal domain of V. cholerae VgrG-3 has bacterial cell wall hydrolase activity (28, 29).Recently, following much effort in the field, a small number of proteins secreted by the T6SS, but not structural components, have been experimentally identified. These proteins are regarded as true secreted substrates of the T6SS, with effector functions in target cells (29–35). For example, antibacterial T6SS-secreted effector proteins with peptidoglycan amidase (cell wall hydrolysis) function, the Type VI amidase effector (Tae) proteins, have been identified in Burkholderia thailandensis (32), P. aeruginosa (31), and Serratia marcescens (30). These Tae proteins play a role in T6SS-mediated antibacterial killing activity and genes encoding four families of Tae protein have been widely identified in other bacteria with T6SSs (32). T6SS-secreted effector proteins which are not peptidoglycan hydrolases have also been reported, including Tse2 secreted by P. aeruginosa, which acts in the bacterial cytoplasm (31), and the VasX and TseL proteins secreted by the V. cholerae T6SS, which are suggested to target membrane lipids (29, 34, 35). In the case of antibacterial T6SSs, the secreting bacterial cells are protected from their own T6SS effector proteins by specific immunity proteins (29–32, 35). However, given the large number of T6SSs in different bacterial species and their apparent ability to secrete multiple substrates, experimentally identified T6-secreted effector proteins still remain surprisingly scarce.Here we report the identification of multiple T6SS-secreted effector proteins in S. marcescens. S. marcescens is an opportunistic pathogen, for example causing ocular infections, nosocomial septicemia and pneumonia (36). Previously, we have identified a T6SS in S. marcescens Db10, which targets and efficiently kills other bacterial cells and plays a role in antibacterial competition (37). We have recently demonstrated that this T6SS secretes two antibacterial effectors, the Tae4 homologs Ssp1 and Ssp2, with cognate immunity proteins Rap1a and Rap2a (30).In this work, we report the analysis of the T6SS-dependent secretome of S. marcescens by label-free quantitation (LFQ) mass spectrometry and describe the identification and characterization of four novel T6SS-secreted effector proteins. These were confirmed as antibacterial toxins and specific immunity proteins were identified. Additionally, this global secretomic analysis, in combination with genetic and phosphoproteomic analyses, demonstrated that a post-translational phosphorylation system influences the ability of the S. marcescens T6SS to secrete effector proteins. Although this system uses homologs of the P. aeruginosa PpkA, PppA and Fha components, the circumstances and impact of Fha phosphorylation were shown to vary between organisms.     

Delineation of a Carcinogenic Helicobacter pylori Proteome

Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet only a fraction of infected persons ever develop cancer. The extensive genetic diversity inherent to this pathogen has precluded comprehensive analyses of constituents that mediate carcinogenesis. We previously reported that in vivo adaptation of a non-carcinogenic H. pylori strain endowed the output derivative with the ability to induce adenocarcinoma, providing a unique opportunity to identify proteins selectively expressed by an oncogenic H. pylori strain. Using a global proteomics DIGE/MS approach, a novel missense mutation of the flagellar protein FlaA was identified that affects structure and function of this virulence-related organelle. Among 25 additional differentially abundant proteins, this approach also identified new proteins previously unassociated with gastric cancer, generating a profile of H. pylori proteins to use in vaccine development and for screening persons infected with strains most likely to induce severe disease.Helicobacter pylori is a Gram-negative bacterial species that selectively colonizes gastric epithelium and induces an inflammatory response within the stomach that persists for decades (1, 2). Biological costs incurred by the long term relationship between H. pylori and humans include an increased risk for distal gastric adenocarcinoma (3–8), and eradication of this pathogen significantly decreases cancer risk among infected individuals without premalignant lesions (9). However, only a fraction of colonized persons ever develop neoplasia, and enhanced cancer risk is related to H. pylori strain differences, inflammatory responses governed by host genetic diversity, and/or specific interactions between host and microbial determinants (10).H. pylori strains are remarkably diverse (11–15), and the genetic composition of strains can change over time within an individual colonized stomach (16, 17). Despite this diversity, several genetic loci have been identified that augment disease risk. The cag pathogenicity island encodes a type IV bacterial secretion system, and the product of the terminal gene in this island, CagA, is translocated into host epithelial cells by the cag secretion system following adherence (18–20). Within the host cell, CagA undergoes Src- and Abl-dependent tyrosine phosphorylation (21) and activates the eukaryotic phosphatase SHP-2, leading to dephosphorylation of host cell proteins and cellular morphological changes (19–21). CagA also dysregulates β-catenin signaling (22, 23) and apical-junctional complexes (24), events linked to increased cell motility and oncogenic transformation in several models (25, 26). Another H. pylori constituent linked to gastric cancer is the cytotoxin VacA, encoded by the gene vacA, which is present in virtually all H. pylori strains (27). In vitro, VacA induces the formation of intracellular vacuoles (27) and can induce apoptosis (28), and vacuolating activity is significantly associated with the presence of the cag pathogenicity island (3).Approximately 20% of H. pylori bind to gastric epithelial cells in vivo (29), and sequence analysis has revealed that the H. pylori genome contains an unusually high number of ORFs relative to its genome size that are predicted to encode outer membrane proteins (15). BabA, a member of a family of highly conserved outer membrane proteins and encoded by the strain-specific gene babA2, binds the Lewis^b histo-blood group antigen on gastric epithelial cells (30, 31), and H. pylori babA2⁺ strains are associated with an increased risk for gastric cancer (30). However, not all persons infected with cag⁺ babA2⁺ toxigenic strains develop gastric cancer, indicating that additional H. pylori constituents are important in carcinogenesis.We recently identified a strain of H. pylori, 7.13, that reproducibly induces gastric cancer in two rodent models of gastritis, Mongolian gerbils and hypergastrinemic INS-GAS mice (22). This strain was derived via in vivo adaptation of a clinical H. pylori strain, B128, which induces inflammation, but not cancer, in rodent gastric mucosa. The oncogenic 7.13 phenotype is not due to an enhanced ability of strain 7.13 to colonize as there were no significant differences in gastric colonization density or efficiency between strains B128 and 7.13 as assessed by either quantitative culture or histology. However, carcinogenic strain 7.13 binds more avidly to gastric epithelial cells in vitro than does strain B128, suggesting that the two strains may variably express different outer membrane proteins.To define proteins that may mediate the development of H. pylori-induced gastric cancer, we performed two-dimensional (2D)¹ DIGE coupled with MS to identify differentially abundant membrane-associated and cytosolic proteins from non-carcinogenic H. pylori strain B128 and its carcinogenic derivative, strain 7.13 (22). DIGE/MS is a well established proteomics technology based on conventional 2D gel protein separations whereby prelabeling samples with spectrally resolvable fluorescent dyes and multiplexing samples onto a series of gels that contain a mixture of all experimental samples (internal standard) provide quantitative data on abundance changes for thousands of intact proteins from multiple experimental conditions, each measured in replicate for statistical confidence (32–36). Techniques including DIGE/MS have recently been utilized to robustly define differences in protein abundance profiles between bacterial strains and to compare expression patterns of proteins harvested from bacteria maintained under different growth conditions (37, 38).Utilizing DIGE/MS, we detected and identified 26 proteins with statistically significant differences between strains B128 and 7.13, including a novel cysteine-to-arginine mutation in the H. pylori flagellar protein FlaA. We demonstrate that this FlaA mutation results in structural and functional aberrations. Application of this technique to two genetically related bacterial strains that induce distinct phenotypes also identified several novel candidate H. pylori virulence factors, providing a framework for studies targeting the pathogenesis of microbially induced cancer.