TraT Protein Research Articles

MP02-11 CHARACTERIZATION OF COMPLEMENT RESISTANCE FACTOR TRAT ENCODED IN DRUG RESISTANCE PLASMIDS OF UROPATHOGENIC ESCHERICHIA COLI

You have accessJournal of UrologyCME1 May 2022MP02-11 CHARACTERIZATION OF COMPLEMENT RESISTANCE FACTOR TRAT ENCODED IN DRUG RESISTANCE PLASMIDS OF UROPATHOGENIC ESCHERICHIA COLI Chae Hee Lim, Aryana Bhati, Katelyn Lott, Jazmyn Whitfield, John Land, and Tatyana Sysoeva Chae Hee LimChae Hee Lim More articles by this author , Aryana BhatiAryana Bhati More articles by this author , Katelyn LottKatelyn Lott More articles by this author , Jazmyn WhitfieldJazmyn Whitfield More articles by this author , John LandJohn Land More articles by this author , and Tatyana SysoevaTatyana Sysoeva More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002514.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The TraT protein is a multifunctional virulence factor that is encoded by conjugative F-like plasmids. A majority of multidrug-resistant uropathogenic Escherichia coli (UPEC) carry F-like plasmids and thus encode for this TraT factor. In non-pathogenic E. coli, TraT is a highly expressed outer membrane lipoprotein that protects the bacterial cell from excessive rounds of plasmid transfer, from a few bacteriophages, and from the host innate immune response via serum complement killing. Our objectives in this study were (1) to measure the protective effect of TraT in UPEC background and (2) to establish whether extracellular TraT domain is sufficient to provide serum resistance. METHODS: We used conjugation to transfer the model F’ plasmid in different E. coli backgrounds. Recombinant extracellular soluble domain TraT33-244 was purified from BL21(DE3) E. coli strain using Ni-affinity chromatography. Exponentially growing E. coli cultures with and without traT-encoding plasmids or the purified TraT domain were subjected to different doses of blood serum for 2 hours at 37°C. Viable cells were quantified as colony forming units by plating serial dilutions at 0 and 2 hours after addition of serum. RESULTS: The traT-encoding plasmid provides variable level of protection that depends on serum type, bacterial cells, time, and ratios of bacteria and serum, and can reach several orders of magnitude. We have developed a method of expression and purification of soluble TraT33-244 protein and showed that this extracellular domain forms stable oligomers in solution with five-to-six subunits. Testing this purified extracellular domain in serum resistance assays revealed that it is insufficient to exhibit the full protective action of TraT. CONCLUSIONS: In this study, we measured the protective effect of traT-encoding plasmid F’ in model E. coli strains, which displayed strong but variable serum resistance. The extracellular soluble domain of TraT protein oligomerizes, but does not strongly protect against serum killing when added to bacterial cells in trans. These results suggest that membrane localization, N-terminus, or lipidation itself are important for function of the TraT factor. Overall, this study sets the stage for further investigation into the mechanism of how TraT assists UPEC bacteria in evading innate immune responses within the host organism. Source of Funding: The University of Alabama in Huntsville startup funds to Tatyana Sysoeva © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e14 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chae Hee Lim More articles by this author Aryana Bhati More articles by this author Katelyn Lott More articles by this author Jazmyn Whitfield More articles by this author John Land More articles by this author Tatyana Sysoeva More articles by this author Expand All Advertisement PDF DownloadLoading ...

A vaccine conjugate of 'ISCAR' immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants.

TraT protein, known as ISCAR (= Immunostimulatory Carrier), is one of a family of integral membrane proteins (Imps) of Escherichia coli representing powerful carrier molecules which when injected into experimental animals generate substantial antibody and T proliferative responses to molecules conjugated to it. We extend these findings to show that ISCAR functions to stimulate Th1- and Th2-type responses, including specific cytotoxic T cells and tumour protection. We report here that by conjugating to ISCAR a 19mer peptide containing linear B epitopes, a T helper (Th) epitope, and a H-2b-restricted T cytotoxic (CTL) epitope of E7 protein of human papillomavirus type 16 (HPV16), and immunizing C57B1/6 (H-2b) mice, we elicited (i) specific IgG2a and IgG1 antibodies; (ii) IL-2 and IL-4 production by specifically recalled lymph node cells in vitro; (iii) cytotoxic T lymphocytes which specifically killed both E7 peptide-pulsed, and whole E7 gene-transfected tumour target cells; and (iv) in vivo protection against an E7 gene-transfected tumour cell inoculum. These findings have implications for the design of vaccines to stimulate immune responses to endogenously processed target antigens (e.g. tumour-associated antigens) without the unwanted side effects of oil-based adjuvants. In addition they support the case for a E7-targeted therapeutic vaccine for HPV-associated human cervical cancer.

Alterations in T cell signal transduction by M. leprae antigens is associated with downregulation of second messengers PKC, calcium, calcineurin, MAPK and various transcription factors in leprosy patients

Mycobacterium leprae, the causative agent of leprosy, challenges host defense mechanism by impairing the signal transduction of T cells which leads to downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. In this study we sought to identify how soluble forms of M. leprae antigen(s) or particulate (liposome) delivery of the same antigens with two immunomodulators Murabutide and T cell peptide of Trat protein influence the transcription of IL-2 gene in anergic T cells of lepromatous patients. It was demonstrated that MLCwA/ManLAM stimulated cells of BL/LL patients showed defects in both jun-NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activities there by resulting in decreased AP-1 activity. Additionally these cells showed reduced calcium levels, PKC activity and calcineurin (CN) activity. This led to impaired nuclear translocation of NFκB and NFAT in these patients. In contrast, when same M. leprae antigen(s) were incorporated with the two immunomodulators in liposomal form, increased transcription of IL-2 gene was observed especially in BL/LL patients which appears to be due to, at least in part, to increased expression of AP-1 Fos and Jun family members, NFκB and NFAT1 proteins. The increased expression of these transcription factors correlated with increased ERK/JNK, PKC and CN activities in these patients. Since activation of ERK/JNK/PKC kinases and CN phosphatase are required for stimulation of IL-2 transcription, these data provide a molecular explanation for the block in IL-2 production by M. leprae antigens. Thus the above study revealed suppression of all the three distinct biochemical pathways, viz. Ca-CN-NFAT pathway, PKC-NF-κB pathway, and MAPK-AP-1 pathway by M. leprae antigen(s) in anergized T cells of lepromatous patients which were activated by liposomal delivery of M. leprae antigens containing the two immunomodulators leading to optimal induction of IL-2 gene expression, which was required for the activation, and proliferation of T cells in lepromatous patients

Mycobacterial formulation in liposomes showed decreased expression of CD95/CD95L and caspase activity in T cells of leprosy patients

Leprosy, a debilitating disease shows manifestations ranging from strong cell mediated immune (CMI) response in tuberculoid form (BT/TT) or weak CMI with immunological anergy in lepromatous form (BL/LL). Repeated stimulation with mycobacterial antigen causes activation induced cell death (apoptosis) or anergy in these patients. Previous studies showed in vitro T cell proliferation and increased Th1 response with liposomal (particulate) presentation of mycobacterial antigens with immunomodulators (MDP analog, murabutide and T cell peptide of Trat protein). In the present study, the role of caspases; expression of apoptotic markers, CD95 and CD95L in T cells of leprosy patients and the effect of particulate formulations in the reversal of apoptosis in BT/TT and BL/LL patient group was studied. Caspase 8/3 activity was maximum (p < 0.01) in BL/LL patients in constitutive state, which was followed by decreased activity in BT/TT and normal subjects. Caspase 8/3 activity increased significantly (p < 0.05) from constitutive state after 5 days of antigen stimulation (inducible state). Both the antigens were equally potent in inducing the apoptosis in leprosy patients. A decreased caspase activity using the particulate formulations of both antigens was observed in BL/LL group. The expression of CD95 and CD95L in T cells of leprosy patients increased significantly after 5 days of antigen stimulation. However, % expression of apoptotic markers by T cells of BL/LL patients decreased significantly (p < 0.05) when the particulate formulations were used. To summarize, the particulate formulations of antigens with immunomodulators slowed down the apoptosis and hence T cell anergy in leprosy by stimulating the cells to proliferate.

Expression of costimulatory molecules (CD80, CD86, CD28, CD152), accessory molecules (TCR αβ, TCR γδ) and T cell lineage molecules (CD4+, CD8+) in PBMC of leprosy patients using Mycobacterium leprae antigen (MLCWA) with murabutide and T cell peptide of Trat protein

In leprosy, cell-mediated immunity (CMI) is more significant than humoral response to eliminate intracellular pathogen. T cell defect is a common feature in lepromatous leprosy (LL) patients as compared to tuberculoid type (TT) patients. For efficient initiation of CD4+, T cell response requires T cell receptor (TCR) activation and costimulation provided by molecules on antigen-presenting cells (APC) and their counter receptors on T cells. In our previous study, the defective T cell function in LL patients was restored to a proliferating state with the release of TH1 type cytokines using mycobacterial antigen(s) with two immunomodulators (Murabutide (MDP-BE) and T cell epitope of Trat protein of Escherichia coli) by presenting the antigen in particulate form in vitro to PBMC derived from leprosy patients. This observation prompted us to study the expression of the costimulatory molecules (CD80, CD86, CD28, CD152), other accessory molecules (TCR αβ/γδ) and T cell lineage molecules (CD4+ and CD8+) during constitutive and activated state of peripheral blood mononuclear cells (PBMC) derived from normal and leprosy individuals using different formulations of Mycobacterium leprae total cell wall antigen (MLCWA), Trat and MDP-BE using flow cytometric analysis. An increased surface expression of CD80, CD86 and CD28 but decreased CD152 expression was observed when PBMC of normal, BT/TT (tuberculoid) and BL/LL (lepromatous) patients were stimulated in vitro with MLCWA+MDP-BE+Trat peptide using liposomal mode of antigen delivery, while opposite results were obtained with the antigen alone. Antibody inhibition study using antihuman CD80 or CD86 completely abolished the T cell lymphoproliferation, thereby reconfirming the importance of these costimulatory molecules during T cell activation/differentiation. Though the liposome-entrapped antigen formulation has no effect on expression of αβ/γδ T cell receptor, the constitutive levels of TCR γδ were high in lepromatous patients. Thus, TCR bearing γδ appears to have a negligible regulatory role in peripheral blood of leprosy patients. The percentage of cells positive for CD4+ are increased in inducible state in all the three groups, while CD8+-positive cells were decreased in LL patients, thereby reconfirming the fact that priming of CD4+ cells are necessary for producing final effector functions. Lastly, intracellular cytokine staining experiment indicated that CD4+ cells are the major producers of IFN-γ but not NK cells. The study highlights the reversal of T cell anergy especially in lepromatous patients through the modulation of costimulatory molecule expression under the influence of Th1 cytokines, i.e., IL-2 and IFNγ.

Expression of Foreign Antigens on the Surface of Escherichia coli by Fusion to the Outer Membrane Protein TraT

The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence of traT, was amplified and obtained by PCR. This sequence was then subcloned downstream of the tac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane of E. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as an E. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.

Expression of foreign antigens on the surface of Escherichia coli by fusion to the outer membrane protein traT.

The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence of traT, was amplified and obtained by PCR. This sequence was then subcloned downstream of the tac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane of E. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as an E. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.

Surface exclusion specificity of the TraT lipoprotein is determined by single alterations in a five-amino-acid region of the protein.

The TraT protein is a highly cell-surface-exposed lipoprotein specified by F-like plasmids that confers serum resistance and blocks the conjugative transfer of plasmids to cells bearing identical or closely related plasmids, a process known as surface exclusion. The TraT protein specified by the antibiotic-resistance plasmid R6-5 was purified to apparent homogeneity. When added to mating mixtures, TraT blocked the transfer of plasmids belonging to Surface Exclusion Group IV (Sfx IV) but had no significant effect on the transfer of plasmids belonging to other groups. Additionally, the purified protein has a protective effect on bacterial cells incubated in serum, indicating that it does not have to be located on the cell surface to mediate serum resistance. To localize regions of the protein that were responsible for surface exclusion specificity, the amino acid sequence of the TraT protein specified by CoIB2-K98 (Sfx II) was determined by cloning and sequencing of the corresponding gene. Comparison of the derived sequence with those of the F and R100-1 proteins indicated that surface exclusion specificity of TraT is determined by single alterations in a five-amino-acid region (residues 116-120). This was confirmed by segment swapping experiments in which the specificity of the R6-5 TraT protein (Sfx IV) was switched to that of the CoIB2-K98 protein (Sfx II). Our results suggest that the region defined by residues 116-120 is located on the external face of the outer membrane and interacts specifically with the donor cell in surface exclusion.

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Abstract Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.

New T-cell epitope derived from the TraT protein of Escherichia coli; T-lymphocyte epitope useful as carrier in vaccine to enhance antibody production and stimulate strong cell-mediated immunity

New T-cell epitope derived from the TraT protein of Escherichia coli; T-lymphocyte epitope useful as carrier in vaccine to enhance antibody production and stimulate strong cell-mediated immunity

Outer membrane permeability of Escherichia coli K12: isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant

We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485-510 kb) whereas sipC and sipD both map to 82 min (3850-3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.

TraT lipoprotein, a plasmid-specified mediator of interactions between gram-negative bacteria and their environment

The TraT protein is a cell-surface-exposed, outer membrane lipoprotein specified by large, usually conjugative, F-like plasmids. Two biological activities have been associated with the protein: (i) prevention of self-mating of cells carrying identical or closely related conjugative plasmids, by blocking the formation of stable mating aggregates; and (ii) resistance to the bactericidal activities of serum, possibly by inhibiting the correct assembly or efficient functioning of the terminal membrane attack complex of complement. The protein therefore interacts not only with components of the outer membrane but also with specific external agents. In conjugative plasmids the traT gene lies within the region necessary for the conjugal transfer of DNA (tra), although its expression is not necessarily dependent on the expression of other tra genes. Recently, however, the gene has been discovered in isolation from other tra genes in nonconjugative virulence-associated plasmids, providing further evidence that the TraT protein may have a role in pathogenesis. The nucleotide sequences of several traT genes have been determined, and comparison of the corresponding amino acid sequences suggests that a central region of five amino acid residues flanked by hydrophobic domains determines the specificity of the protein in surface exclusion. Additionally, studies of mutants with different amino acid alterations within the hydrophobic domains have shown that insertion of charged residues disrupts normal outer membrane integrity. This review considers our current knowledge of the distribution, structure, and biological role(s) of the protein. Recent applications of the protein in studies of the unusual permeability properties of the outer membrane and for the transport of foreign antigenic determinants to the bacterial cell surface are also discussed.

Presentation of foreign antigenic determinants at the bacterial cell surface using the TraT lipoprotein

The TraT protein is an oligomeric outer membrane lipoprotein, specified by plasmids of the IncF group, that is very highly exposed at the bacterial cell surface. We have investigated the feasibility of using the protein as a carrier of foreign antigenic determinants by genetic insertion of the C3 epitope of type 1 poliovirus into defined sites in the protein. Several of the hybrid proteins constructed had features characteristic of the native protein and one in particular retained the ability to function in surface exclusion and genetic suppression assays as well as to assemble into oligomers. Our results suggest that the TraT protein can be used to transport and present foreign antigenic determinants at the cell surface.

The pYV plasmid of Yersinia encodes a lipoprotein, YlpA, related to TraT

A series of lipoproteins was detected in the membrane fraction of Yersinia enterocolitica W227, a typical strain from serotype O:9. At least two of them, YlpA and YlpB, are encoded by the pYV plasmid. The sequence of ylpA reveals the presence of a typical lipoprotein signal peptide. The mature YlpA protein would be 223 residues long with a calculated molecular weight of 23798 for the proteic moiety of the molecule. YlpA shares 88% identical residues with the TraT protein encoded by plasmid pED208, 80% identity with TraT proteins encoded by plasmids R100 and F, and 77% identity with the TraT protein encoded by the virulence plasmid of Salmonella typhimurium. The ylpA gene hybridized with the pYV plasmid of Yersinia pseudotuberculosis, suggesting that this gene is conserved among Yersinia spp. The production of YlpA is controlled by virF and only occurs at 37 degrees C in the absence of Ca2+ ions. This co-regulation with the yop genes suggests that ylpA is a virulence determinant. However, mutations in ylpA clearly affect neither the resistance to human serum nor the virulence for intravenously inoculated mice.

The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure–function relationships in the TraT protein

The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti-C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild-type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.

TraT lipoprotein, a plasmid-specified mediator of interactions between gram-negative bacteria and their environment.

The TraT protein is a cell-surface-exposed, outer membrane lipoprotein specified by large, usually conjugative, F-like plasmids. Two biological activities have been associated with the protein: (i) prevention of self-mating of cells carrying identical or closely related conjugative plasmids, by blocking the formation of stable mating aggregates; and (ii) resistance to the bactericidal activities of serum, possibly by inhibiting the correct assembly or efficient functioning of the terminal membrane attack complex of complement. The protein therefore interacts not only with components of the outer membrane but also with specific external agents. In conjugative plasmids the traT gene lies within the region necessary for the conjugal transfer of DNA (tra), although its expression is not necessarily dependent on the expression of other tra genes. Recently, however, the gene has been discovered in isolation from other tra genes in nonconjugative virulence-associated plasmids, providing further evidence that the TraT protein may have a role in pathogenesis. The nucleotide sequences of several traT genes have been determined, and comparison of the corresponding amino acid sequences suggests that a central region of five amino acid residues flanked by hydrophobic domains determines the specificity of the protein in surface exclusion. Additionally, studies of mutants with different amino acid alterations within the hydrophobic domains have shown that insertion of charged residues disrupts normal outer membrane integrity. This review considers our current knowledge of the distribution, structure, and biological role(s) of the protein. Recent applications of the protein in studies of the unusual permeability properties of the outer membrane and for the transport of foreign antigenic determinants to the bacterial cell surface are also discussed.

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid

The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.

The role of the traT gene of the Salmonella typhimurium virulence plasmid for serum resistance and growth within liver macrophages

The role of the traT gene of the Salmonella typhimurium virulence plasmid for the serum resistance of the bacteria and their growth within mouse liver macrophages was investigated. The gene product, the TraT protein, increased the serum resistance in E. coli HB101, which naturally does not carry traT. It also contributed to the serum tolerance of S. typhimurium. The capacity of an isogenic S. typhimurium TML strain triplet, differing in their ability to express TraT and in the quality of the traT gene expressed, to grow in vivo in the mouse liver indicated that, although TraT was dispensable for the net growth of the bacteria within the liver, the expression of a mutated traT gene reduced the growth rate. The traT gene was mapped on the virulence plasmid outside previously defined virulence determinants suggesting that other regions of the virulence plasmid are mainly responsible for the growth within mouse liver macrophages in S. typhimurium.

The outer membrane permeability mutation of the virulence-associated plasmid ofSalmonella typhimuriumis located in atraT-like gene

The SS-A mutation carried by the virulence-as-associated plasmid of Salmonella typhimurium results in increased outer membrane permeability to hydrophobic compounds. A 7.8-kilobase pair BamHI-SalI fragment containing the SS-A mutation was cloned from the virulence-associated plasmid into the cloning vector pACYC184. The cloned DNA segment hybridized with a radioactive probed prepared from the traT gene of R6-5. A similar DNA fragment, cloned from the wild-type virulence-associated plasmid, complemented the SS-A mutant phenotype. Both clones produced a protein that immunologically resembled the R6-5 TraT protein; however, the protein produced by the SS-A containing clone appeared truncated by approximately Mr 1000 indicating an alteration in the primary structure or processing of the protein. We conclude that the mutation producing the SS-A phenotype has occured in a traT-like gene of the Salmonella plasmid.