Hemolysin-negative Mutant Research Articles

Identification and Characterization of a Repeat-in-Toxin Gene Cluster in Vibrio anguillarum

Vibrio anguillarum is the causative agent of vibriosis in fish. Hemolysins of V. anguillarum have been considered virulence factors during infection. One hemolysin gene, vah1, has been previously identified but does not account for all hemolytic activity. The mini-Tn10Km mutagenesis performed with a vah1 mutant resulted in a hemolysin-negative mutant. The region surrounding the mutation was cloned and sequenced, revealing a putative rtx operon with six genes (rtxACHBDE), where rtxA encodes an exotoxin, rtxC encodes an RtxA activator, rtxH encodes a conserved hypothetical protein, and rtxBDE encode the ABC transporters. Single mutations in rtx genes did not result in a hemolysin-negative phenotype. However, strains containing a mutation in vah1 and a mutation in an rtx gene resulted in a hemolysin-negative mutant, demonstrating that the rtx operon is a second hemolysin gene cluster in V. anguillarum M93Sm. Reverse transcription-PCR analysis revealed that the rtxC and rtxA genes are cotranscribed, as are the rtxBDE genes. Additionally, Vah1 and RtxA each have cytotoxic activity against Atlantic salmon kidney (ASK) cells. Single mutations in vah1 or rtxA attenuate the cytotoxicity of V. anguillarum M93Sm. A vah1 rtxA double mutant is no longer cytotoxic. Moreover, Vah1 and RtxA each have a distinct cytotoxic effect on ASK cells, Vah1 causes cell vacuolation, and RtxA causes cell rounding. Finally, wild-type and mutant strains were tested for virulence in juvenile Atlantic salmon. Only strains containing an rtxA mutation had reduced virulence, suggesting that RtxA is a major virulence factor for V. anguillarum.

Serratia marcescens internalization and replication in human bladder epithelial cells.

BackgroundSerratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains.MethodsElektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production.ResultsWe identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production.ConclusionThe results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections.

Prevalence of, antibody response to, and immunity induced by Haemophilus ducreyi hemolysin.

Haemophilus ducreyi, the etiologic agent of chancroid, a genital ulcer disease, produces a cell-associated hemolysin whose role in virulence is not well defined. Hemolysin is encoded by two genes, hhdA and hhdB, which, based on their homology to Serratia marcescens shlA and shlB genes, are believed to encode the hemolysin structural protein and a protein required for secretion and modification of this protein, respectively. In this study, we determined the prevalence and expression of the hemolysin genes in 90 H. ducreyi isolates obtained from diverse geographic locations from 1952 to 1996 and found that all strains contained DNA homologous to the hhdB and hhdA genes. In addition, all strains expressed a hemolytic activity. We also determined that hemolysin is expressed in vivo and is immunogenic, as indicated by the induction of antibodies to hemolysin in both the primate and rabbit disease models as well as in human patients with naturally acquired chancroid. Wild-type strain 35000 and isogenic hemolysin-negative mutants showed no difference in lesion development in the temperature-dependent rabbit model. However, immunization of rabbits with the purified hemolysin protein reduced the recovery of wild-type H. ducreyi, but not hemolysin-negative mutants, from lesions. Our study indicates that hemolysin is a possible candidate for vaccine development due to its immunogenicity, expression in vitro and in vivo by most, if not all, strains, and the effect of immunization on reducing the recovery of viable H. ducreyi in experimental disease in rabbits.

Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection.

To examine the role of flagella in pathogenesis of urinary tract infection caused by Proteus mirabilis, we constructed a nonmotile, nonswarming flagellum mutant of strain WPM111 (an hpmA hemolysin mutant of strain BA6163, chosen because of its lack of in vitro cytotoxicity in renal epithelial cell internalization studies). A nonpolar mutation was introduced into the flaD gene, which encodes the flagellar cap protein. This mutation does not affect the synthesis of flagellin but rather prevents the assembly of an intact flagellar filament. In in vitro assays, the genetically characterized nonmotile mutant was found to be internalized by cultured human renal proximal tubular epithelial cells in numbers less than 1% of those of the flagellated parent strain. Internalization of the nonmotile mutant was increased significantly (14- to 21-fold) by centrifugation onto the monolayer. To assess virulence in vivo, CBA mice were challenged transurethrally with 10(7) CFU of P. mirabilis BA6163 (wild type) (n = 16), WPM111 (hpmA mutant) (n = 46), or BB2401 (hmpA flaD mutant) (n = 46). Differences in quantitative cultures between the parent strain and the hemolysin-negative mutant were not significant. However, the hpmA flaD mutant was recovered in numbers approximately 100-fold lower than those of the hmpA mutant or the wild-type parent strain and thus was clearly attenuated. We conclude that while hemolysin does not significantly influence virulence, flagella contribute significantly to the ability of P. mirabilis to colonize the urinary tract and cause acute pyelonephritis in an experimental model of ascending urinary tract infection.

Virulence of Culture Filtrate from Heat-Injured and Repaired Listeria Strains: Assay on Bovine Mammary Epithelial (MAC-T) Cells

The cytotoxic effect of culture filtrates from healthy, heat-stressed, and repaired Listeria monocytogenes and L. innocua on the bovine mammary epithelial cell line MAC-T was examined. Culture filtrates were collected from Listeria spp. following treatments which included: (i) 18 h of growth of Listeria at 37°C; (ii) sublethal heat treatment at 56°C for 50 minutes; (iii) repair of the injured cells at 37°C for 7 h; (iv) growth of repaired bacterial cells at 37°C for 36 h; and (v) heat injury at 56°C for 50 min of the cell population obtained after the initial repair and growth. Strains chosen for study included two genetic mutants of L. monocytogenes: a hemolysin-negative mutant, CNL 85/162 (Hly−) and a hemolysin-positive revertant, CNL 85/163 (Hly+). Culture filtrates obtained from Hly− bacteria did not prevent adhesion of the mammary epithelial cells and slightly stimulated their growth. In contrast, culture filtrates from Hly+ bacteria grown for 18 h significantly reduced the ability of MAC-T cells to adhere to the cell culture dishes, prevented the growth of those cells that were attached to the dishes, and caused cell death. Supernatants from Hly+ and Hly− following injury and during repair had no lethal effect on MAC-T cells. The effects of culture medium obtained after growth of the repaired Listeria cells on MAC-T cells were similar to those recorded for medium from the first 18 h growth for both strains, indicating that cells regain virulence potential once they have repaired and reinitiated growth. Culture filtrates obtained from L. monocytogenes Scott A showed results similar to those of Hly+, decreasing adherence and growth of MAC-T cells, while L. innocua culture filtrate had no adverse effect. The results of these experiments suggest that when injured, L. monocytogenes does not demonstrate adverse effects towards MAC-T cells. Once repair is completed and the listeria are growing, activity towards MAC-T cells is restored.

Binding to and killing of human renal epithelial cells by hemolytic P-fimbriated E. coli

Acute pyelonephritis is a common invasive infection frequently caused by E. coli that possess P-fimbriae and secrete hemolysin. We have examined the role of P fimbriae and hemolysin in the killing of putative target cells of acute pyelonephritis, that is, human renal epithelial cells (HRPTEC). Cultures of HRPTEC were overlaid with (1) a prototypic pyelonephritogenic E. coli (CFT073) which expresses both P fimbriae and hemolysin; (2) its hemolysin-negative isogenic mutant (CFT073hlyD::TnphoA); or (3) a prototypic nonpyelonephritogenic fecal E. coli (FN414) which is negative for both P fimbriae and hemolysin. CFT073 and CFT073hlyD::TnphoA but not FN414 adhered to HRPTEC, as demonstrated by electron microscopy and direct counting. Adherence was diminished by antisera directed against P fimbriae and by a monoclonal antibody recognizing the epithelial receptor for P fimbriae. CFT073 was significantly more cytolethal for HRPTEC than its hemolysin-negative mutant. The bacteria-free filtrate of CFT073 was both hemolytic and cytolethal whereas that of CFT073hyD::TnphoA was not hemolytic and was significantly less cytolethal. Finally, we demonstrated that CFT073 passed through monolayers of HRPTEC at a higher rate than CFT073hlyD::TnphoA, indicating that hemolysin damages HRPTEC, facilitating passage of bacteria through the epithelial barrier. With HRPTEC and a pyelonephritogenic strain of E. coli we have reproduced in vitro bacterial attachment and toxin delivery by P fimbriae and hemolysin, factors epidemiologically associated with acute pyelonephritis in patients.

Reduced virulence of Serpulina hyodysenteriae hemolysin-negative mutants in pigs and their potential to protect pigs against challenge with a virulent strain

The role of the Serpulina hyodysenteriae hemolysin encoded by the tlyA gene in the pathogenesis of swine dysentery (SD) was studied. tlyA mutants of two S. hyodysenteriae strains (B204 and C5) were tested for virulence in pigs. None of the animals developed SD. However, after infection with wild-type strain B204 or C5, the incidence of SD was 100 or 60%, respectively. Thus, the tlyA-encoded hemolysin of S. hyodysenteriae is an important virulence factor in SD. The potential of these mutants to protect pigs against challenge with a virulent S. hyodysenteriae strain was also studied. After challenge with wild-type strain B204, 50% of pigs previously inoculated with the B204 tlyA mutant were protected, whereas all control pigs contracted SD. None of the pigs previously inoculated with the C5 tlyA mutant developed SD upon challenge with wild-type strain B204, whereas 40% of the control pigs developed SD in this experiment. Thus, previous colonization with S. hyodysenteriae tlyA mutants in pigs provides partial protection against challenge with a virulent S. hyodysenteriae strain.

Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes.

The gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen. The only known property of L. monocytogenes which has been shown to be involved in virulence is a hemolysin, listeriolysin (J. L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986; S. Kathariou, P. Metz, H. Hof, and W. Goebel, J. Bacteriol. 169:1291-1297, 1987). Using our previously obtained transposon Tn916-induced hemolysin-negative mutants of L. monocytogenes Sv1/2a (Mackaness strain), we demonstrated that the loss of hemolysin reduced significantly the rate of survival of the bacteria in mouse peritoneal macrophages but did not reduce their uptake. It was further shown that virulent L. monocytogenes strains could invade the mouse embryo fibroblast 3T6 cell line, i.e., mammalian cells which are nonprofessional phagocytes. This uptake was inhibited by cytochalasin B and hence seems to be accomplished by parasite-induced endocytosis. Hemolysin was not essential for this step. Strains of other Listeria species could not efficiently penetrate the 3T6 cells.

Characterization of hemolysin in extracellular products of Pseudomonas cepacia.

Pseudomonas cepacia is recognized as an opportunistic pathogen in immunocompromised patients. We screened 120 strains of P. cepacia isolated from clinical specimens for production of extracellular products. About 70% of these strains produced lipase, protease, and lecithinase, but only 4% produced hemolysin. A hemolysin produced by P. cepacia JN106 was characterized. The hemolysin was most active against human erythrocytes. Horse, sheep, chicken, and rabbit erythrocytes were also susceptible. The hemolysin was heat labile and was inhibited by sterols but was not activated by 2-mercaptoethanol and dithiothreitol. Four hemolysin-negative mutants obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment produced the other extracellular products. A 58-kilobase-pair plasmid found in the parent strain was also found in the mutant strains, suggesting that the hemolysin gene resides on the chromosome.

A role for hemolysin in Escherichia coli-induced inflammation in granulocytopenic rabbits.

Inflammation induced in the skin of granulocytopenic rabbits by Escherichia coli was examined. Protein exudation and platelet deposition in lesions were measured with 125I-labeled albumin and 111In-labeled platelets. In granulocytopenic rabbits 10(4)-10(7) live serum-resistant E. coli induced protein exudation and platelet deposition beginning at 3 hr and then progressing over the next 24 hr to much higher levels than in normal rabbits. These responses were associated with interstitial edema and progressive venous thrombosis in the absence of leukocytes; no such reactions were observed in normal rabbits. No reactions were induced in granulocytopenic rabbits by killed E. coli. Of six E. coli strains tested, all three hemolytic strains induced lesions with four to five times more thrombosis (platelet deposition) than did nonhemolytic strains. Two hemolysin-negative mutants lost most of their thrombogenic activity. All three hemolytic strains had cell-associated hemolysin, but only one of these elaborated appreciable free, filterable hemolysin as well.

Multiple copies of hemolysin genes and associated sequences in the chromosomes of uropathogenic Escherichia coli strains.

The O6 serogroup Escherichia coli strain 536 carries two hemolysin (hly) determinants integrated into the chromosome. The two hly determinants are not completely identical, either functionally or structurally, as demonstrated by spontaneous deletion mutants carrying only one of them and by cloning each of the two determinants separately into cosmid vectors. Each hly determinant is independently deleted at a frequency of 10(-4), leading to variants which exhibit similar levels of internal hemolysin but different amounts of secreted hemolysin. The two hly determinants were also identified in the O4 E. coli strain 519. The three E. coli strains 251, 764, and 768, which belong to the serogroup O18, and the O4 strain 367 harbor a single chromosomal hly determinant, as demonstrated by hybridization with hly-gene-specific probes. However, a hybridization probe derived from a sequence adjacent to the hlyC-proximal end of the plasmid pHly 152-encoded hly determinant hybridizes with several additional chromosomal bands in hemolytic O18 and O6 E. coli strains and even in E. coli K-12. The size of the probe causing the multiple hybridization suggests a 1,500- to 1,800-base pair sequence directly flanking hlyC. Spontaneous hemolysin-negative mutants were isolated from strains 764 and 768, which had lost the entire hly determinant but retained all copies of the hlyC-associated sequence.2+.

Spontaneous deletions and flanking regions of the chromosomally inherited hemolysin determinant of an Escherichia coli O6 strain.

The hemolytic Escherichia coli strain 536 (O6) propagates spontaneous hemolysin-negative mutants at relatively high rates (10(-3) to 10(-4)). One type of mutant (type I) lacks both secreted (external) and periplasmic (internal) hemolysin activity (Hlyex-/Hlyin-) and in addition shows no mannose-resistant hemagglutination (Mrh-), whereas the other type (type II) is Hlyex-/Hlyin+ and Mrh+. The genetic determinants for hemolysin production (hly) and for mannose-resistant hemagglutination (mrh) of this strain are located on the chromosome. Hybridization experiments with DNA probes specific for various parts of the hly determinant reveal that mutants of type I have lost the total hly determinant, whereas those of type II lack only part of the hlyB that is essential for transport of hemolysin across the outer membrane. Using a probe that contains the end sequence of the plasmid pHly152-encoded hly determinant (adjacent to hlyB), we determined that a related sequence flanks also the hlyB-distal end of the chromosomal hly determinant of E. coli 536. In addition several other similar or even identical sequences are found in the vicinity of the hlyC- and the hlyB-distal ends of both the chromosomal and the plasmid hly determinants.