Elena V. Kozlova

Chronic herpesvirus reactivation occurs in aging.

The aged immune system is characterized by clonal expansions of CD8+ T cells of which a substantial portion are directed against Epstein-Barr virus (EBV) and cytomegalovirus (CMV). It is unknown if these expansions represent increased viral reactivation or simply reflect an accumulation over time. We investigated herpesvirus reactivation in young and old subjects co-infected with CMV and EBV. Using molecular and serological techniques, we found significant increases in both the frequency and magnitude of EBV and CMV reactivation in elderly subjects. CMV DNA was frequently detected in the urine of elderly subjects; EBV load in peripheral blood was also significantly increased. Notably, EBV DNA in plasma was detected in a majority of the elderly subjects which was supported by frequent transcription of late structural genes. Furthermore, CD8+ T cells specific for EBV structural antigens were detected in samples from the elderly. Samples from our younger control group were negative for EBV DNA in plasma, CMV DNA in urine, expression of structural transcripts, and lacked CD8+ T cells specific for EBV structural antigens. These findings indicate that the aged immune system is no longer able to control EBV and CMV reactivation that could now be characterized as chronic instead of latent.

Role of Various Enterotoxins in Aeromonas hydrophila-Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity

ABSTRACT Three enterotoxins from the Aeromonas hydrophila diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (act)-positive or -negative background strain of A. hydrophila. We developed two single knockouts with truncation in either the heat-labile (alt) or the heat-stable (ast) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i) alt and ast, (ii) act and alt, and (iii) act and ast genes; and a triple-knockout mutant with truncation in all three genes, act, alt, and ast. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type A. hydrophila; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected A. hydrophila mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in A. hydrophila-induced gastroenteritis in a mouse model with the greatest contribution from the cytotoxic enterotoxin Act, followed by the Alt and Ast cytotonic enterotoxins.

N-Acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout Delta ahyRI mutant. Protease production was substantially reduced in the Delta ahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the Delta ahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the Delta ahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the Delta ahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the Delta ahyRI/ahyR(+) complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

Molecular Characterization of a Glucose-Inhibited Division Gene, gidA, That Regulates Cytotoxic Enterotoxin of Aeromonas hydrophila

ABSTRACT By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

Characterization of Aeromonas hydrophila Wound Pathotypes by Comparative Genomic and Functional Analyses of Virulence Genes

ABSTRACT Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966T, and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966T. The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966T and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity. IMPORTANCE Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms. Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms.

Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila.

A diarrheal isolate SSU of Aeromonas hydrophila produces a cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. Our laboratory has characterized from the above Aeromonas strain, in addition to Act, the type 3- and T6-secretion systems and their effectors, as well as the genes shown to modulate the production of AI-1-like autoinducers, N-acylhomoserine lactones (AHLs) involved in quorum sensing (QS). In this study, we demonstrated the presence of an S-ribosylhomocysteinase (LuxS)-based autoinducer (AI)-2 QS system in A. hydrophila SSU and its contribution to bacterial virulence. The luxS isogenic mutant of A. hydrophila, which we prepared by marker exchange mutagenesis, showed an alteration in the dynamics and architecture of the biofilm formation, a decrease in the motility of the bacterium, and an enhanced virulence in the septicemic mouse model. Moreover, these effects of the mutation could be complemented. Enhanced production of the biofilm exopolysaccharide and filaments in the mutant strain were presumably the major causes of the observed phenotype. Our earlier studies indicated that the wild-type A. hydrophila with overproduction of DNA adenine methyltransferase (Dam) had significantly reduced motility, greater hemolytic activity associated with Act, and an enhanced ability to produce AI-1 lactones. Furthermore, such a Dam-overproducing strain was not lethal to mice. On the contrary, the luxS mutant with Dam overproduction showed an increased motility and had no effect on lactone production. In addition, the Dam-overproducing luxS mutant strain was not altered in its ability to induce lethality in a mouse model of infection when compared to the parental strain which overproduced Dam. We suggested that an altered gene expression in the luxS mutant of A. hydrophila SSU, as it related to biofilm formation and virulence, might be linked with the interruption of the bacterial metabolic pathway, specifically of methionine synthesis.

The two-component QseBC signalling system regulates in vitro and in vivo virulence of Aeromonas hydrophila

We recently demonstrated that the N-acyl-homoserine lactone [autoinducer (AI)-1] and LuxS (AI-2)-based quorum-sensing (QS) systems exerted positive and negative regulation, respectively, on the virulence of a diarrhoeal isolate SSU of Aeromonas hydrophila. However, the role of a newly identified, two-component-based QseBC QS system in the regulation of bacterial virulence in general is not well understood, with only a limited number of studies showing its function in bacterial pathogenesis. In this report, we identified and characterized the QseBC QS system in A. hydrophila SSU and found that, as was the case with enterohaemorrhagic Escherichia coli, the open reading frames for the qseB (the response regulator) and qseC (the sensor histidine kinase) genes overlapped by 4 bp at the ATGA motif. Our data provide evidence that deletion of the qseB gene from A. hydrophila resulted in attenuation of bacterial virulence in a septicaemic mouse model of infection and diminished swimming and swarming motility, and the mutant bacteria formed denser biofilms compared with those from the parental strain of A. hydrophila. The decrease in the virulence of the A. hydrophila ΔqseB mutant correlated with reduced production of protease and the cytotoxic enterotoxin, which has associated haemolytic activity. The swimming and swarming motility, haemolytic activity, protease production and biofilm formation were restored in the qseBC-complemented strain to a level similar to that of the wild-type A. hydrophila SSU. Our study is the first, to our knowledge, to report a functional QseBC QS system in A. hydrophila which may be linked to AI-1 and AI-2 QS systems in modulating bacterial virulence, possibly through the cyclic diguanosine monophosphate.

Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks

ABSTRACT We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutants growth dropped slightly during the late phase at 37°C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 105 to 5 × 105 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 108 CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

Quorum sensing and c-di-GMP-dependent alterations in gene transcripts and virulence-associated phenotypes in a clinical isolate of Aeromonas hydrophila

Recently, we demonstrated that the LuxS-based quorum sensing (QS) system (AI-2) negatively regulated the virulence of a diarrheal isolate SSU of Aeromonas hydrophila, while the ahyRI-based (AI-1) N-acyl-homoserine lactone system was a positive regulator of bacterial virulence. Thus, these QS systems had opposing effects on modulating biofilm formation and bacterial motility in vitro models and in vivo virulence in a speticemic mouse model of infection. In this study, we linked these two QS systems with the bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) in the regulation of virulence in A. hydrophila SSU. To accomplish this, we examined the effect of overproducing a protein with GGDEF domain, which increases c-di-GMP levels in bacteria, on the phenotype and transcriptional profiling of genes involved in biofilm formation and bacterial motility in wild-type (WT) versus its QS null mutants. We provided evidence that c-di-GMP overproduction dramatically enhanced biofilm formation and reduced motility of the WT A. hydrophila SSU, which was equitable with that of the ΔluxS mutant. On the contrary, the ∆ahyRI mutant exhibited only a marginal increase in the biofilm formation with no effect on motility when c-di-GMP was overproduced. Overall, our data indicated that c-di-GMP overproduction modulated transcriptional levels of genes involved in biofilm formation and motility phenotype in A. hydrophila SSU in a QS-dependent manner, involving both AI-1 and AI-2 systems.

Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila.

ABSTRACT The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.