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Infection and Immunity, October 2005, p. 6446-6457, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6446-6457.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

Jian Sha,{dagger} Lakshmi Pillai,{dagger} Amin A. Fadl, Cristi L. Galindo, Tatiana E. Erova, and Ashok K. Chopra*

Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-1070

Received 25 April 2005/ Returned for modification 15 June 2005/ Accepted 8 July 2005


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ABSTRACT
 
Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.


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INTRODUCTION
 
Aeromonas species are emerging human pathogens that cause a wide array of diseases, such as gastroenteritis, wound infections, and septicemia (49, 67). One of the most potent virulence factors of Aeromonas species is the type II-secreted cytotoxic enterotoxin (Act), which possesses hemolytic, cytotoxic, and enterotoxic activities and is lethal in nanogram quantities when injected into mice (12, 82).

Recently, genes for a type III secretion system (TTSS) were identified in fish isolates of Aeromonas spp. (4, 5, 6, 73, 80, 84). The TTSS enables many pathogenic gram-negative bacteria to secrete and inject pathogenicity proteins (effectors) into the cytosol of eukaryotic cells via needle-like structures called needle complexes or injectisomes. Overall, type III secretion systems have been grouped into five major families based on phylogenetic analysis of three highly conserved proteins (i.e., homologs of YscN, YscV, and YscC in Yersinia enterocolitica), and, thus far, more than 30 different effectors have been described among several bacteria (23, 38). These effectors have been shown to possess multiple biological functions, such as cytoskeletal alterations and activation of intracellular signaling cascades within the host cells (23, 38, 79). The TTSS was initially discovered in human and plant pathogens; however, the presence of a TTSS was recently reported in some endosymbionts and invertebrate pathogens (15, 23, 25). It appears that one of the functions of TTSS might be to allow bacteria to establish transkingdom cell-cell communications, thus facilitating persistence and replication of the organism in the host (23).

Quorum sensing (QS), another type of cell-cell communication that also exists in many gram-negative bacteria (8, 18, 26, 50), involves the production of extracellular signaling molecules, called autoinducers (AIs), which proportionally increase as the bacteria grow. When a critical threshold concentration of autoinducer is reached, the bacteria detect the signal, coordinately alter gene expression, and thus respond as a group (34). The phenomenon of QS was first characterized in the bioluminescent bacterium Vibrio fischeri, which involves a two-component LuxI/LuxR system, with LuxI functioning as an autoinducer synthase (18). Other autoinducer synthases, such as LuxM, HdtS, or LuxS, have also been identified in different bacteria (18, 30, 34, 44, 50, 63, 75). Interestingly, there are no sequence homologies between the LuxI family, LuxM, or HdtS autoinducer synthases. However, they all synthesize AI-1-like autoinducers, N-acylhomoserine lactones (AHLs), which constitute lactone-based QS systems (22, 34). These autoinducers bind to either a transcriptional activator, such as a member of the LuxR family (18, 22), or a biosensor (sensor kinases of the phosphorylation cascade), such as LuxN, which results in the regulation of expression of virulence genes in certain bacteria (22, 34). The examples of these QS-regulated virulence factors are the TTSSs in Vibrio harveyi and Vibrio parahaemolyticus (34), the ExoS effector protein in Pseudomonas aeruginosa (36), and the biofilm formation in Aeromonas hydrophila (46).

All of the AHLs described to date contain an invariant homoserine lactone moiety and a highly variable fatty acyl group, which ranges from 4 to 18 carbon atoms in length (88). The homoserine lactone moiety is derived from S-adenosylmethionine, while the acyl chains are derived from acyl-acyl carrier proteins (54, 57, 59, 88). Another type of autoinducer synthase (LuxS) catalyzes S-ribosylhomocysteine to produce a five-carbon furanone (an AI-2 autoinducer) that binds to a biosensor, such as LuxPQ (sensor kinases of the phosphorylation cascade, like LuxN) in V. harveyi and V. parahaemolyticus (22, 34). The AI-2 autoinducer has been proposed to be specific for interspecies communication in bacteria (22, 63, 75). Unlike AI-1, which is an autoinducer only in gram-negative bacteria, AI-2 serves as a QS signal molecule in both gram-negative and gram-positive bacteria (22, 39). It has been reported that AI-2-mediated QS regulates virulence factors in certain bacteria, such as toxin production in Clostridium perfringens, and expression of pagA, lef, and cya genes that code for protective antigen, lethal factor, and edema factor, respectively, in Bacillus anthracis. As a result, novel therapies have been proposed for anthrax using inhibitors to AI-2 (22, 39, 56).

These two different QS systems (AI-1 and AI-2) can coexist in the same bacterium, such as the LuxM/LuxN and the LuxS/LuxPQ systems of V. harveyi and V. parahaemolyticus (34). On the other hand, some bacteria harbor more than one lactone-based QS system, like LasI/LasR and RhlI/RhlR in P. aeruginosa or LuxI/LuxR and AinS/AinR (the homolog of LuxM/LuxN) in V. fischeri (17, 30, 43, 45). Likewise, the homolog of LuxI/LuxR, designated AhyI/AhyR, was detected in an A. hydrophila A1 strain, while AsaI/AsaR was identified in Aeromonas salmonicida NCIMB 1102 (76). Both AhyI and AsaI synthesize a major AHL, N-(butanoyl)-L-homoserine lactone (BHL), and a minor AHL, N-hexanoyl-L-homoserine lactone (HHL) (76). The role of QS in regulating biofilm maturation and extracellular protease production was reported in A. hydrophila (46, 77). However, other investigators found no correlation between extracellular protease production and QS in this pathogen (81).

Two biosensor strains, namely, Agrobacterium tumefaciens A136 and Chromobacterium violaceum CV026, have been successfully used for detecting lactones in clinical isolates of A. hydrophila and P. aeruginosa (87). C. violaceum CV026 was used as a biosensor to detect AHL with N-acyl side chains of four to eight carbons, especially BHL (48). A. tumefaciens A136 was used as another biosensor (28), which is extremely sensitive to the 3-oxo derivatives with N-acyl chain length from six to 12 carbons, including N-(3-oxododecanoyl)-L-homoserine lactone (69). The observation that most strains of A. hydrophila and P. aeruginosa produced AHLs that could be detected by both CV026 and A136 revealed that the majority of the isolates of these bacteria produced multiple AHLs (87).

In this study, we identified a TTSS gene cluster from the clinical isolate SSU of A. hydrophila. The aopB gene of the TTSS is involved in the formation of a needle complex and has been reported to be essential in inducing the TTSS-associated cytotoxicity in in vitro cell culture models. Further, the aopB mutants were noted to be less virulent in the animal models (4, 80, 84). Likewise, by preparing an act mutant, we demonstrated that the type II-secreted Act also plays an important role in Aeromonas infections (82). Therefore, we inactivated the aopB gene from the WT A. hydrophila SSU, as well as from a previously characterized act-minus mutant of A. hydrophila SSU (82), to evaluate their contribution in the pathogenesis of Aeromonas infections. By using aopB, act, and act/aopB mutants of A. hydrophila, we demonstrated a role for the TTSS and Act in host cell cytotoxicity and animal lethality. Furthermore, we found a unique correlation of the TTSS and Act with AHL production in this bacterium. The effects of TTSS and Act deletion on cytotoxicity and lactone production could be complemented.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. A. hydrophila SSU, its rifampin-resistant (Rifr) derivative, and an act isogenic mutant in which the toxin gene was interrupted by a kanamycin resistance (Kmr) gene cassette were previously described (67, 82). Vectors pBluescript and pBR322 were used for cloning, and plasmid pBRaopB, which contained the coding region of the A. hydrophila aopB gene, was used for complementation. A suicide vector, pDMS197, with a conditional R6K origin of replication (ori), a levansucrase gene (sacB) from Bacillus subtilis, and a tetracycline resistance (Tcr) gene was used for homologous recombination (19). The plasmid pHP45{Omega}, containing a streptomycin and spectinomycin resistance (Sm/Spr) gene cassette, was employed as a selective marker for generating isogenic mutants (67). Ampicillin, tetracycline, kanamycin, spectinomycin, and streptomycin were used at concentrations of 100, 15, 50, 25, and 25 µg/ml, respectively, in Luria-Bertani (LB) medium or agar plates. Rifampin was utilized at a concentration of 40 µg/ml for bacterial growth and 300 µg/ml during conjugation experiments. Chromosomal DNA was isolated using a QIAamp DNA minikit, and digested plasmid DNA or DNA fragments from agarose gels were purified using a QIAprep Miniprep kit (QIAGEN, Inc., Valencia, CA). The bacterial strains and plasmids used in this study are listed in Table 1.


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  		TABLE 1. Strains and plasmids used in this study

Cloning and DNA sequence analysis of the A. hydrophila SSU TTSS. Among different genes that constitute a TTSS, sequences of some genes (e.g., yscV from Yersinia species and its homologs in other bacteria) are highly conserved (23). Therefore, primers (Table 2) were synthesized, based on the yscV gene of Y. enterocolitica TTSS (70). Next, PCR amplification of the corresponding gene (ascV) from the genome of A. hydrophila was performed under the following conditions: 96°C for 5 min (denaturation), followed by 35 cycles of 96°C for 1 min, 65°C for 1 min, and then 72°C for 1 min. The final extension was performed at 72°C for 7 min. The amplified ascV DNA fragment (2,166 bp) was sequenced, and additional primers were subsequently designed based on the sequence of the ascV gene, which allowed us to perform further chromosomal sequencing and to obtain a partial DNA sequence of the TTSS of A. hydrophila SSU.


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  		TABLE 2. Sequences of the primers used in this study

To obtain the entire DNA sequence of the TTSS of A. hydrophila, we simultaneously prepared a fosmid library (titer, ~1 x 106 CFU/ml) of A. hydrophila SSU, using a pEpiFOS-5 fosmid vector (chloramphenicol resistance; Epicentre, Madison, WI), sheared and blunt-ended chromosomal DNA, MaxPlax lambda packaging extracts, and Escherichia coli EPI100 plating cells. The library was prepared following the manufacturer's instructions. The fosmid library was screened with an [{alpha}-32P]dCTP-labeled ascV gene (2,166-bp) probe of A. hydrophila using colony blot hybridization (68). Briefly, the fosmid library was plated directly onto the nylon filters (Gibco BRL, Gaithersburg, MD) and placed on the surface of LB agar plates containing 12.5 µg/ml chloramphenicol. Each filter with 150 to 200 colonies was prehybridized (2 h) and then hybridized (using 32P-labeled ascV gene probe) in Quikhyb solution (Stratagene, La Jolla, CA) at 68°C for 3 h. The membranes were washed twice at 68°C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) plus 0.1% sodium dodecyl sulfate (SDS) for 20 min and then twice in 1x SSC plus 0.1% SDS for 20 min at 68°C (68). The blots were exposed to X-ray film at –70°C for 2 to 12 h. Three rounds of colony blot hybridization were performed to ensure purity of the clones. Finally, DNA isolated from positive colonies was used as a template for DNA sequencing.

All of the DNA sequencing was performed with an automated DNA sequencer, 373XL (Applied Biosystems, Inc., Foster City, CA), in the Protein Chemistry Core Laboratory at the University of Texas Medical Branch, Galveston. The DNA sequence data were analyzed and compared with the databases using an online BCM Search Launcher (Baylor College of Medicine Human Genome Sequencing Center, Houston, TX) and the Clustal W program (Supercomputer Laboratory, Institute for Chemical Research, Kyoto University, Kyoto, Japan).

Generation and characterization of aopB and act/aopB mutants of A. hydrophila SSU. Two pairs of primers were synthesized that amplified the upstream and downstream flanking sequences to the aopB gene (Table 2). The resulting 604- and 783-bp DNA fragments were ligated together through a common EcoRI site and cloned into a pBluescript vector at KpnI/XbaI restriction enzyme sites, resulting in a recombinant plasmid, pBlueUD. A streptomycin-spectinomycin (Sm/Spr) gene cassette flanked by an EcoRI site from plasmid pHP45{Omega} was inserted at the EcoRI site of pBlueUD to generate a recombinant plasmid, pBlueUDSm/Sp. After digestion with KpnI/XbaI restriction enzymes, the DNA fragment from the above plasmid was ligated to a pDMS197 suicide vector at the compatible restriction enzyme sites, and the resulting plasmid (pDMS197UDSm/Sp) was transformed into an E. coli SM10{lambda}pir strain (67). The recombinant E. coli [pDMS197UDSm/Sp] was conjugated with either WT A. hydrophila SSU-R or its act mutant (67, 82). The transconjugants were selected based on resistance to appropriate antibiotics and sucrose and subjected to further analysis (67).

Southern blot analysis. Chromosomal DNA from aopB and act/aopB mutants, as well as from WT A. hydrophila, was isolated, and an aliquot (10 µg) was digested with a PstI restriction enzyme and subjected to 0.8% agarose gel electrophoresis (67). Next, the digested DNA was transferred to a nylon membrane and baked at 80°C for 2 h. Three DNA probes were used for Southern blot analysis. These probes represented the coding region of the aopB gene (1,188 bp that was PCR amplified using specific primers [Table 2]), a 2.0-kb Sm/Spr gene cassette from plasmid pHP45{Omega} and obtained by BamHI restriction enzyme digestion, and a suicide vector, pDMS197 (6.0 kb) (67). The prehybridization and hybridization conditions were the same as those mentioned above for the colony blot hybridization.

Integrity of the cell membrane. The aopB and act/aopB mutants, as well as the WT A. hydrophila, were grown to an optical density at 600 nm (OD600) of 0.4 to 0.5 and diluted 50-fold, and then various concentrations of Triton X-100 (TX-100; 0.5 to 5%), SDS (0.5 to 2%), and vancomycin (100 to 200 µg/ml) were added. The cultures were incubated at 37°C for 3 h with shaking (180 rpm), and the OD was measured. A 50% reduction in the OD in three independent experiments indicated sensitivity of the cultures to the above-mentioned detergents and the antibiotic. These methods were described in our recent publication (65). We also measured periplasmic RNase I leakage from mutants versus WT A. hydrophila. Briefly, bacterial cells were streaked on LB agar plates containing 1.5% Torula yeast RNA (Sigma, St. Louis, MO) and incubated overnight at 37°C. After incubation, 10% trichloroacetic acid was added onto the plates and the RNase leakage from bacterial cells was examined by measuring the diameter of the clear zones around the bacterial streaks (7).

In vitro binding assay. HT-29 colonic epithelial cells were infected with aopB and act/aopB mutants, as well as with the WT A. hydrophila at a multiplicity of infection (MOI) of 10, and incubated at 4 or 37°C for 1 h. Unbound bacteria were aspirated, cells were washed four times with phosphate-buffered saline (PBS) and lysed with 0.1% TX-100, and various dilutions of the cell lysates were plated onto 1.5% (wt/vol) LB agar plates for determining CFU (20, 65).

Complementation of the A. hydrophila SSU aopB mutant. We used specific primers (Table 2) to amplify the coding region of the aopB gene (1,188 bp). The ends of the DNA fragment were made blunt by using a PCR polishing kit (Stratagene, La Jolla, CA). Subsequently, the blunt-ended DNA fragments were ligated to the pBR322 vector (Amersham Biosciences, Piscataway, NJ) at the ScaI site, which also generated blunt ends. This recombinant plasmid (pBRaopB) was then electroporated into the aopB mutant of A. hydrophila. The pBR322 vector alone was also electroporated into WT A. hydrophila, as well as into the aopB and act mutants (67), and served as a proper negative control. The orientation of the aopB gene in the vector was examined by digestion of the recombinant plasmid with various restriction enzymes (e.g., EcoRI/PstI and HindIII). The aopB gene was under the control of an ampicillin resistance gene promoter in the pBR322 vector.

Cytotoxicity and cell detachment assay. The WT A. hydrophila SSU or act, aopB, or act/aopB mutants and their complemented strains were grown in 3 ml of LB medium in 50-ml disposable tubes and incubated at 37°C overnight with shaking (180 rpm). Supernatants and bacterial cells were separated and collected by centrifugation. The supernatants were filter sterilized (0.22 µm), and the bacterial cells were washed three times with PBS. RAW 264.7 murine macrophages or HT-29 colonic epithelial cell lines (American Type Culture Collection [ATCC], Manassas, VA) were seeded into 96-well plates (1 x 105 cells/well) or chamber slides (1 x 106; Nalge Nunc International, Rochester, NY) and infected with the live bacterial cultures (as prepared above) at an MOI of 10. Host cells were also treated with 5 µl of bacterial culture supernatants. After incubation at 37°C for 2 to 4 h, the tissue culture medium was examined for the release of lactate dehydrogenase (LDH) enzyme using a CytoTox96 kit (Promega, Madison, WI).

Subsequent to bacterial infection, the morphology of the host cells was visualized in 20 to 25 fields by Nomarski differential interference microscopy using a Zeiss UV 510 Meta confocal microscope (Carl Zeiss, Inc., Thornwood, NY) (65). We also calculated the percentage of the detached cells in the monolayer after staining with Giemsa stain. Briefly, the cells in the monolayer were fixed with 70% methanol for 5 min and then stained for 1 to 2 h. Subsequently, the monolayers were dissolved in a RIPA buffer (60) and the plates were shaken gently for 1 h. The blue color that was released from the host cells was measured at 590 nm using a VERSAmax tunable microplate reader (Molecular Devices Corporation, Sunnyvale, CA) (29).

AHL bioassay. AHL levels in culture supernatants of A. hydrophila SSU strains were detected by using the reporter strain A. tumefaciens A136 (ATCC) (86), which lacks the nopaline-type Ti plasmid and, therefore, does not produce its own lactones. However, the presence of a plasmid with traR and a traG:lacZ fusion allows the bacterium to respond to exogenously added lactones. The traR gene of A. tumefaciens resembles luxR of V. fischeri, in that it functions as a quorum-sensing transcriptional activator (27). TraR binds AHLs, forming active homodimers, which, in turn, activate the traG:lacZ reporter gene. Shaw et al. (69) reported that this A. tumefaciens reporter strain was more sensitive than other AHL biosensors and could detect a broad range of AHLs in different bacteria, except for BHL, compared to the C. violaceum CV026 strain (69, 87). However, a more recent study demonstrated that AHLs in the majority of the clinical isolates of A. hydrophila and P. aeruginosa could be detected by using either of the two biosensor strains (A. tumefaciens A136 or C. violaceum CV026) (87). Although BHL was shown to be the major lactone produced in A. hydrophila A1 (76), it is currently unclear which of the different forms of lactones are produced by our clinical isolate SSU of A. hydrophila. We therefore chose to use the A. tumefaciens A136 strain to detect a wide range of characterized and uncharacterized lactones in A. hydrophila SSU. Further, this biosensor strain is commercially available through the ATCC.

The standard protocol using the A. tumefaciens reporter strain was followed. Briefly, overnight-grown A. tumefaciens (in LB medium at 30°C) was diluted to an OD600 of 0.2. An aliquot (2 ml) of diluted A. tumefaciens was mixed with 0.5-ml culture supernatants (filter sterilized) from A. hydrophila (WT, its mutants, and complemented strains) grown to various optical densities (OD600, 0.3 to 1.2) and incubated at 30°C for 5 h (86). This time allowed activation of traR by A. hydrophila lactones and initiation of transcription and amplification of the traG:lacZ reporter gene to produce detectable levels of ß-galactosidase. The pH of the supernatants was monitored, as the AHLs are rapidly inactivated under alkaline conditions (83). The ß-galactosidase activity was then measured according to the method of Miller and reported as Miller units (MU) (52). During the 5-h incubation, the A. tumefaciens grew from an OD600 of 0.2 to one of 0.6, which was still within the exponential growth phase. A. tumefaciens does not produce AHL-inactivating enzymes, such as lactonases, under these conditions (85).

Animal experiments. Groups of 10 Swiss Webster mice (Taconic Farms, CA) were infected by the intraperitoneal route with 5 x 107 to 1 x 108 bacteria (WT or act, aopB, or act/aopB mutants) in accordance with approved animal care protocols. Deaths were recorded for 16 days postinfection. The bacterial doses used represented approximately three 50% lethal doses of WT A. hydrophila (82).

For animal studies, statistical analyses were performed using Fisher's exact test. For all other studies, Student's t test was used. For all of the in vitro and in vivo studies, three independent experiments were performed.

Nucleotide sequence accession number. The DNA sequence of the TTSS operon of A. hydrophila SSU was submitted to GenBank (accession number AY763611).


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RESULTS AND DISCUSSION
 
The TTSS of A. hydrophila SSU. An Aeromonas TTSS was first reported recently in the fish pathogen A. salmonicida (4, 5, 6, 73). We independently amplified, based on the DNA sequence of the Y. enterocolitica yscV gene, the corresponding gene (ascV) from a human diarrheal isolate of A. hydrophila. Then, using chromosomal DNA sequencing and screening of a fosmid library, we obtained the full-length TTSS sequence of A. hydrophila SSU. A total of 750 fosmid clones were screened by colony blot hybridization, and we obtained eight positive clones that reacted with the ascV gene probe. The average length of the inserted DNA fragment in the fosmid clones was 25 kb. The entire TTSS operon contained 26,855 bp, encoding 35 genes, compared to only 20 genes identified for the A. salmonicida TTSS (4) (Table 3).


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  		TABLE 3. Homology of the type III secretion system genes in different Aeromonas strains and Yersinia enterocolitica

While we were sequencing and characterizing the TTSS from the human diarrheal isolate of A. hydrophila, a TTSS was reported for the A. hydrophila fish isolate AH1 (84). However, our SSU clinical isolate contained 10 additional TTSS genes, compared to the AH1 strain, indicating that these 10 additional genes might not exist in the AH1 strain or that the complete operon was not cloned and sequenced from this strain. Conservation was noted in the sequences of the TTSS genes of A. hydrophila SSU, the fish isolate AH1, A. salmonicida, and Y. enterocolitica; however, the sequences differed significantly for certain genes (Table 3). For example, the aopB and aopD genes, the products of which form the TTSS translocation apparatus (4, 84), share only 50 to 53% identity between the A. hydrophila strains AH1 and SSU. These differences might result in a more efficient injection of effector proteins into the respective fish and human host cells.

Interestingly, the acrV gene of A. hydrophila SSU was 147 to 165 bp shorter than that of A. salmonicida or A. hydrophila AH1. DNA sequence alignment revealed several deletions in the middle part of the gene, which resulted in a product that was 41 to 49 amino acids shorter. A similar deletion (41 amino acids) was found in LcrV, the Y. enterocolitica homolog of AcrV. The presence or absence of this stretch of amino acid residues in different Aeromonas strains might alter the function of AcrV in fish versus human hosts and requires further investigation. The TTSS of A. hydrophila SSU possessed two genes, hscY/exsC and ascZ/exsD, that had no homologs in the well-characterized Y. enterocolitica TTSS but were present in the TTSS of P. aeruginosa (24). The exsD gene product is an antitranscriptional activator of ExsA (16), while the exsC gene encodes exoenzyme S synthesis protein C precursor, which is involved in the synthesis of exoenzyme family proteins in P. aeruginosa (24). The hscY/exsC and ascZ/exsD may function similarly in both A. hydrophila SSU and P. aeruginosa.

As our molecular characterization of the A. hydrophila SSU TTSS was near completion, a TTSS gene sequence from a fish isolate of A. hydrophila AH3 was submitted to GenBank (accession number AY528667) and subsequently published (80). The TTSS operon from both the SSU and AH3 strains contained 35 genes (Table 3). The general identities between various genes of the AH3 and SSU TTSS operons were 28 to 89% at the nucleotide level, and at the amino acid level, homologies of 38 to 97% were recorded (Table 3). When we compared the SSU to the AH3 strain, a 49-amino-acid deletion was found in AcrV of strain SSU, which was similar to the deletion in AcrV of A. salmonicida and A. hydrophila AH1. The sequence diversity in the gene encoding a 359-amino-acid-residue-long AscP was maximal between the SSU and AH3 strains (with only 38% homology at the amino acid level) (Table 3). Further comparison of the two strains showed that there were three deletions, of 11, 16, and 28 amino acid residues, in the central part of AscP (between amino acid residues 187 and 228) in SSU.

Recently, a dual function was proposed for YscP, the homolog of AscP, in Y. enterocolitica (1, 40). First, YscP may act as a molecular ruler that determines the length of the TTSS needle (40), whereby the N and C termini of YscP anchor the central potion of the ruler (ruler domain), with the C terminus of YscP being attached to the basal body and the N terminus to the growing tip of the needle (40). Deletion of either the N or C terminus of YscP has been shown to result in a loss of control in needle length. However, deletion of the ruler domain resulted in shorter needles at a rate of 1.9 Å per amino acid residue (40), which could lead to less efficient injection of effectors into host cells (55). We noticed that AscP in the clinical isolate SSU of A. hydrophila was 8 and 48 amino acid residues shorter in the ruler domain than AscP in the fish isolates AH1 and AH3, respectively. This size difference in the central ruler part could potentially lead to a shorter needle in isolate SSU that has less injection efficiency than those in fish isolates AH1 and AH3. The effect on bacterial virulence of a shorter TTSS needle is worth pursuing and will be studied in our future research. Interestingly, AscP in A. salmonicida had the shortest needle of all A. hydrophila strains, with a 39- to 68-amino-acid-residue deletion in its ruler domain.

The second proposed function of YscP is that it may act as a type III secretion substrate specificity switch (T3S4) (1). In this model, the N terminus of YscP is believed to be attached to the growing needle, while the C terminus, containing the T3S4 domain, stays in the secretion apparatus and switches the substrate specificity from YscF to Yops by interacting with YscU in Y. enterocolitica (1). The T3S4 domain of YscP could be replaced by the T3S4 domain of AscP (A. salmonicida) or PscP (P. aeruginosa), indicating that the T3S4 domain is functional in A. salmonicida as well as in P. aeruginosa (1). Although in all of the above-mentioned Aeromonas strains the homology of AscP is only 38 to 57% (Table 3), the T3S4 domain is conserved with a homology of 67 to 85% at the amino acid level, indicating evolutionary relatedness.

Interestingly, DNA sequences flanking the TTSS operon were entirely different in the AH3 and SSU strains, indicating that the chromosomal location of the TTSS differed between A. hydrophila isolates of fish and human origin. More importantly, the TTSS of A. salmonicida was located on a plasmid, while it was found on the chromosome in A. hydrophila isolates (6, 73, 80).

Characterization of aopB and aopB/act mutants of A. hydrophila. Since homologs of AopB and AopD form the translocation apparatuses of other TTSS needle complexes (4, 80, 84), mutations in the aopB and aopD genes could block translocation of A. hydrophila effector proteins. Therefore, we generated aopB deletion mutants in WT and act-minus strains of A. hydrophila via homologous recombination and confirmed their identity by Southern blot analysis. Briefly, digested chromosomal DNA from the aopB mutant reacted with the Sm/Spr gene cassette probe, but not with the probes to the aopB and suicide vector pDMS197, indicating the replacement of the aopB gene with the Sm/Spr gene cassette on the genome of this mutant. Further, an inability of the digested genomic DNA of this mutant to hybridize with the suicide vector probe indicated the loss of the suicide vector as the result of double-crossover homologous recombination (data not shown).

The growth rates of mutants (aopB and act/aopB) and their binding abilities for HT-29 cells were tested and compared to those of the WT bacterium. The mutants behaved very similarly to the WT bacterium in terms of their growth rates and binding to the host cells. Nor was the membrane integrity of the mutants affected, as measured by their ability to grow in the presence of different concentrations of TX-100 and SDS as well as vancomycin. Moreover, the release of periplasmic RNase I was unaltered in the aopB and act/aopB mutants compared to that of the WT A. hydrophila SSU (data not shown). These findings show that the membrane integrity of the mutants remained intact when the genes encoding act and aopB were deleted.

TTSS-associated cytotoxicity of A. hydrophila SSU. To test the function of the TTSS, RAW 264.7 cells were infected with WT A. hydrophila, its various mutants, or complemented strains, and cytotoxicity assays were performed. An LDH release of 18 to 20% was noted (following comparison to the positive control provided in the kit and adjusted to 100% cell lysis) during the initial phase (2 h) of infection with the WT bacterium or act mutant (Fig. 1A). In contrast, infection with aopB and act/aopB mutants resulted in 79% and 71% decreases in LDH release, respectively, compared to that in cells infected with the WT A. hydrophila, and these decreases were statistically significant at P values of 0.0004 and 0.0008 (Fig. 1A). The effects on cytotoxicity at 2 h, resulting from mutation in the aopB gene, could be complemented (Fig. 1A). The increase in cytotoxicity associated with the aopB-complemented strain (aopB+) was significantly higher than that of the aopB mutant (P = 0.0002) but was not significant when the LDH release was compared between the WT and aopB+ strains (P = 0.6) and between WT and the act mutant at 2 h (P = 0.3).



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  		FIG. 1. (A) Cytotoxicity induced by WT A. hydrophila, its various mutants, and complemented strains in RAW 264.7 cells. Macrophages in triplicate wells were infected with live Aeromonas strains at an MOI of 10 for 2 h and 4 h. After incubation, host cell culture supernatants were used to measure LDH release. (B and C) Cytotoxicity induced by overnight-grown bacterial culture supernatants from WT A. hydrophila, its various mutants, and complemented strains in HT-29 (B) and RAW 264.7 (C) cells. Arithmetic means ± standard deviations from three independent experiments were used for plotting the data. Single asterisks denote statistically significant differences (by Student's t test) between various mutant strains and the WT bacterium. A double asterisk denotes a statistically significant difference between 2- and 4-h treatment of host cells with the aopB mutant. A triple asterisk indicates a statistically significant difference between the act mutant and the WT bacterium at 4 h. The actual P values are presented in the text. aopB+ and act+, complemented strains of aopB and act mutants.

More interestingly, after 4 h of infection, macrophage release of LDH was 8.1 times higher with the aopB mutant (with intact act gene) compared to findings after 2 h of infection with this mutant (P = 0.0002) (Fig. 1A). This higher LDH release after 4 h of infection with the aopB mutant may be due to the presence of Act, as no significant increase in LDH release occurred between 2 and 4 h of infection with the act/aopB mutant (P = 0.7), compared with the WT bacterium (P < 0.0001) (Fig. 1A). Act's effect on cytotoxicity at 4 h was almost fully restored when the act mutant was complemented with the act gene (act+), reaching the level of that seen with WT A. hydrophila (P = 0.7). The increase in LDH release between the act mutant and act+ strain at 4 h was statistically significant (P = 0.003).

Compared to that for the WT bacterium, the decrease in LDH release with the act mutant at 4 h was significant (P = 0.004); however, this decrease for the aopB mutant, compared to the rate in WT A. hydrophila, was not significant (P = 0.1), signifying the importance of Act in cytotoxicity. Between 2 and 4 h, the LDH release associated with the WT bacterium increased from 20% to 40% (P = 0.0009), while this increase for the act mutant was not significant (P = 0.06).

These LDH release data indicated that AopB played an important role in cytotoxicity during early (2-h) bacterial-host cell interactions (4, 80, 84), whereas Act's contribution towards cell toxicity became prominent only by 4 h postinfection. This was not surprising, as our previous studies demonstrated that Act could be detected in the culture medium only after 3 to 4 h of growth (66). However, it is also plausible that the TTSS and Act might affect host cells in different ways with different time courses, which will need further investigation.

When HT-29 or RAW 264.7 cells were treated with overnight bacterial culture supernatants from the act mutant, LDH release was inhibited in both types of host cells, compared to when culture supernatants from WT A. hydrophila or aopB mutant were used, with P values of 0.008 for HT-29 and 0.001 for RAW cells, respectively (Fig. 1B and C). Similarly, bacterial culture supernatants from the act/aopB mutant were unable to induce any LDH release in macrophages or HT-29 cells over the same time period of 4 h (P = 0.0005 for RAW and 0.008 for HT-29 cells). However, we could fully restore Act-associated cytotoxicity by complementation (Fig. 1B and C). These data indicated that Act was the only significant cytotoxic factor present in overnight bacterial culture supernatants and, thus, was principally responsible for host cell damage and death, once secreted in sufficient quantities.

Microscopic alteration in the morphology of HT-29 and RAW 264.7 cells infected with the mutant strains of A. hydrophila, as well as the percentage of attached versus detached cells in the monolayer, revealed a trend similar to that observed for LDH release (Fig. 1 and 2). In the cell detachment assay, we recorded the host cells that remained attached to the wells of the plates after being stained with Giemsa stain. At 2 h postinfection, treatment with aopB or act/aopB mutant resulted in 95 to 98% fewer macrophages and intestinal epithelial cells that detached from the monolayers, compared to those infected with WT or act mutant bacteria (30 to 35%) (P < 0.0001) (Fig. 2C). Uninfected cells were designated as control. In concordance with the LDH release data at 4 h postinfection, a rapid host cell detachment (42%) was noted with the aopB mutant, reaching to the level seen with the WT A. hydrophila (51%). The difference in the cell detachment between WT and aopB mutant at 4 h was not statistically significant (P = 0.07) (Fig. 2C). However, the host cells infected with the act/aopB mutant exhibited similar cell detachment patterns at 2 and at 4 h, indicating the significance of Act in cell toxicity when it is produced by the bacterium (Fig. 2C). We also noted that the decrease in cell detachment with the act mutant was statistically significant (P = 0.001) at 4 h compared to the WT-infected host cells. Likewise, the increase in cell detachment with the aopB mutant at 2 versus 4 h was also significant (P = 0.0003). The cell detachment assay results coincided with the morphology of the host cells as depicted in Fig. 2A and B. The HT-29 cells infected with either the WT or the act mutant were highly vacuolated and flattened after 2 h of infection. However, host cells infected with the aopB or act/aopB mutants showed normal morphology (Fig. 2A). Likewise, macrophages infected with the WT or the act mutant were rounded, while host cells infected with aopB and act/aopB mutants exhibited a normal morphology (Fig. 2B). These alterations in morphology are indicative of cytotoxicity, which we confirmed by the cell detachment and LDH release assays.



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  		FIG. 2. Alteration of cell morphology induced by WT A. hydrophila and its various mutants in HT-29 (A) and RAW 264.7 (B) cells. Host cells were infected at an MOI of 10 and incubated for 2 h. Cells were then washed in PBS, fixed with 4% paraformaldehyde, and examined by Nomarski differential interference microscopy. Noninfected cells (control) and those infected with the aopB or act/aopB mutants exhibited normal cell morphology, while cell morphology was significantly altered (highly vacuolated and flattened [HT-29] or rounded [macrophages]) in cells infected with the WT or the act mutant. The experiment was performed in triplicate, and representative pictures are shown. For the cell detachment assay (C), HT-29 or RAW 264.7 cells were infected with the above-mentioned bacteria at an MOI of 10 for 2 and 4 h. The percentage of cells detached from the monolayers was calculated by Giemsa staining and solubilization of macrophages/HT-29 cells to release the blue color, the intensity of which was measured at 590 nm as described in Materials and Methods. Single asterisks denote statistically significant differences (by Student's t test) between various mutant strains and the WT bacterium. A double asterisk denotes a statistically significant difference between 2- and 4-h treatment of host cells with the aopB mutant. A triple asterisk indicates a statistically significant difference between the act mutant and the WT bacterium at 4 h.

We do not know the precise mechanism by which AopB induces cell death of macrophages and intestinal epithelial cells. AopB homologs from other bacterial pathogens, such as Yersinia pseudotuberculosis (YopB), Salmonella enterica serovar Typhimurium (SipB), Shigella flexneri (IpaB), P. aeruginosa (PopB), and Bordetella bronchiseptica (BopB), have been shown to function as the TTSS translocon (32, 38, 42, 58, 74). The translocon forms a pore in the host membrane, allowing translocation of the effectors into the cytosol of eukaryotic cells, which leads to cytotoxicity (32, 33, 38, 42, 58, 74). Pore formation mediates the contact-dependent hemolytic activity in these bacteria and is an essential step for the translocation of effectors through the TTSS (14, 32, 35, 42, 51). Further, IpaB, SipB, and BopB could act as effectors and translocate themselves into the host cell as well (11, 13, 42). It has been reported that IpaB and SipB induce apoptosis through ICE (interleukin-1ß converting enzyme) or caspase 1 in professional phagocytes (10, 11, 78, 89); however, recent studies have implicated that the induced cytotoxicity (especially by SipB) has more features of necrosis and hence it was termed programmed necrosis (3, 31). BopB is essential in Bordetella TTSS-induced, caspase-1-independent necrosis (42, 72). However, it is not clear whether it is directly involved in the induction of necrosis (42). In contrast, YopB does not translocate itself (37). However, it could activate proinflammatory signaling responses in Yersinia-infected epithelial cells (61, 79). The exact role of AopB in Aeromonas infections is not known and will be investigated in our future studies.

In A. salmonicida, the ADP ribosyltransferase toxin AexT (homolog of P. aeruginosa ExoT/S) was reported to function as a TTSS effector protein that caused cell death, and the role of AopB as the TTSS translocon was proposed (2, 4). Based on dot blot analysis, 78 to 86% of A. hydrophila clinical isolates were found to be positive when hybridized with the aexT gene probe (9). However, it remains to be seen whether an AexT homolog is present in our clinical isolate.

In our in vivo studies, we noted that 100% of the animals infected with WT A. hydrophila SSU at doses of 5 x 107 to 1 x 108 organisms died within 48 h (Fig. 3). However, only 50 to 60% (P = 0.02 to 0.03 compared to WT bacteria) of the animals died when inoculated with the act or aopB mutant of A. hydrophila SSU. In contrast, 90% of the animals (P = 0.001 compared to WT bacteria) that were infected with the same doses of the act/aopB mutant survived over a test period of 16 days (Fig. 4), which indicates that the presence of both the TTSS and Act was crucial for Aeromonas-mediated lethality in mice.



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  		FIG. 3. Mutations in both the act and aopB genes rendered A. hydrophila SSU avirulent in a mouse model. Swiss Webster mice (n = 10 per group) were injected intraperitoneally with three 50% lethal doses of indicated mutants and the WT A. hydrophila SSU and monitored for death over a period of 16 days. The data were statistically analyzed using Fisher's exact test. Three independent experiments were performed, and data from a typical experiment are shown. Asterisks denote statistically significant differences between act/aopB mutant and WT bacteria. The actual P values are presented in the text.



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  		FIG. 4. Effect on lactone production by the TTSS and Act of A. hydrophila SSU. WT A. hydrophila, its various mutants, and complemented strains were grown to various densities (OD600 of 0.3 to 1.2). The cultures were centrifuged, and the supernatants were measured for ß-galactosidase activity (presented in Miller units), indicative of AHL production, using an A. tumefaciens ß-galactosidase reporter strain. Three to five independent experiments were performed, and the arithmetic mean ± deviation was plotted. Single asterisks denote statistically significant differences (by Student's t test) in lactone production between WT and its various mutant derivatives at an OD of 1.2. A double asterisk denotes a statistically significant difference in lactone production in WT and its act mutant at ODs of 0.9 and 1.2. The increase in lactone production by the complemented strains was significantly higher than that in the corresponding mutants but was statistically insignificant compared to lactone production in the WT bacterium. Strains designated act+ and aopB+ indicate complementation of the corresponding mutants. The actual P values are presented in the text. Compl, complementation.

QS and the TTSS. Our studies indicated that both Act and TTSS contributed to the virulence of A. hydrophila. Recently, studies have shown that QS regulates TTSS in several bacteria. In enterohemorrhagic E. coli, the QS system LuxS/LuxPQ regulates the locus of the enterocyte effacement operon that, in turn, controls the TTSS (71). On the other hand, in V. harveyi and V. parahaemolyticus, QS was shown to down-regulate the TTSS genes vopD, vopN, and vopB (Vibrio outer membrane proteins D, N, and B) at high cell density (34).

In P. aeruginosa, two QS systems have been identified: LasI/LasR and RhlI/RhlR, both of which represent LuxI/LuxR homologs. While LasI synthesizes BHL, RhlI produces N-(3-oxododecanoyl)-L-homoserine lactone. Mutations in rhlI or rhlR resulted in the up-regulation of exoS (encoding the TTSS effector ExoS) expression during biofilm formation in P. aeruginosa. This exoS up-regulation phenomenon in the RhlI mutant was repressed by adding BHL, indicating a negative regulatory effect of RhlR/BHL on exoS expression (36). In addition to cell density, QS itself can also be regulated by a variety of factors, such as RpoS ({sigma}S) and RpoN ({sigma}54), which control the formation of flagella and pili and the production of exotoxin A in P. aeruginosa (64).

These studies led us to speculate whether deletion of a major virulence factor gene (act) or the TTSS aopB gene would alter QS autoinducer (lactone) production in A. hydrophila SSU. As shown in Fig. 4, WT A. hydrophila lactone production was significantly increased by high cell density, most notably between an OD of 0.9 and one of 1.2 (an increase in MU from 254 to 951 [P = 0.0003]), which is similar to the general trend observed for other gram-negative bacteria (21). Likewise, for the act mutant, there was a statistically significant (P = 0.03) increase in lactone production (from 331 to 642 MU) when the bacterium grew from an OD of 0.9 to one of 1.2. However, lactone production was reduced by 32% in the act mutant compared to that in WT A. hydrophila at an OD of 1.2 (P = 0.02) (Fig. 4).

More importantly, we did not observe any significant increase in lactone production by the aopB mutant grown to an OD of 0.9 or 1.2. Overall, there was a 64% reduction in lactone production between WT A. hydrophila and the aopB mutant at an OD of 1.2 (P = 0.0001). Lactone production was reduced even further in the act/aopB mutant, with a 92% decrease in lactone production compared to that of WT A. hydrophila when their growth reached an OD of 1.2 (P < 0.0001) (Fig. 4), possibly suggesting an additive effect of the act and aopB single gene deletions. We were able to restore QS in the act (P = 0.04) and aopB (P = 0.006) mutants after complementation (designated as act+ and aopB+), as measured by ß-galactosidase activity (Fig. 4). Statistically, no significant difference was noted in lactone production between WT A. hydrophila and the act+ strain (P = 0.2) and between WT bacteria and the aopB+ strain (P = 0.5).

Lactone-based QS exists in many gram-negative bacteria, and microorganisms often produce more than one type of AHL (26, 50, 76). Identification of AHLs in bacterial culture supernatants or in purified form requires appropriate bioassay strains. Although a variety of bioassay strains have been constructed (88), most of them can detect only a narrow range of AHLs. For example, C. violaceum reporter strain CV026 cannot detect any of the 3-hydroxy derivatives and lacks sensitivity to most 3-oxo derivatives (8, 47). LuxR-based reporters detect most of the 3-oxo and alkanoyl standards but not 3-hydroxy forms of lactones (8, 62). It is possible that, in addition to BHL and HHL, which are identified by using C. violaceum reporter strain CV026 (76), Aeromonas species could also produce other types of AHLs. There was, in fact, an unidentified spot on the thin-layer chromatography plates that migrated between HHL and BHL in A. salmonicida (76).

Recently, Kirke et al. (41) reported that AhyR regulates AhyI production in a growth phase-dependent manner in A. hydrophila, as AhyI accumulated in the exponential phase and degraded in the stationary phase (41). However, the AhyI levels were sustained in the stationary phase of the ahyR mutant. Thus, the degradation of AhyI might be due to the production of a protease, or alternatively, a second QS circuit exists in which another LuxR homolog controls the production of AhyI in the ahyR mutant (41). Nevertheless, the reporter strain A. tumefaciens A136 was used successfully by other investigators (87), and in this study, to detect AHL production by A. hydrophila, indicating that not only BHL but also other AHLs could play an important role in the QS system of A. hydrophila.

In conclusion, we showed, for the first time, a correlation between the presence of a TTSS, Act, and lactone production, in a diarrheal isolate (SSU) of A. hydrophila. It is plausible that QS ensures that a sufficient number of bacteria are present to coordinate the expression of a virulence-associated gene(s) that could overwhelm host defenses. In addition, lactone-based quorum sensors are almost always integrated into other regulatory circuitry (26). This effectively expands the range of environmental signals that influence target gene expression beyond population density (26). As we previously reported, Act is a type II-secreted cytotoxic enterotoxin (12), and the expression of the act gene was affected by different environmental stimuli, including its regulation through the fur regulatory circuitry (68). Act production increased as bacterial density increased (66), and thus, it is plausible that act gene expression is also under the control of QS, which is a topic worth pursuing in the future. Complex regulatory networks exist in bacteria and are required for bacteria to assemble secretion machineries (e.g., type II and III secretion systems), as well as to produce and secrete proteins. It is therefore possible that the bacteria could "sense" and subsequently "react" to dysfunction of the components in these complex networks, thereby "shutting off" the production of signaling molecule lactones in an attempt to conserve "bacterial energy." This scenario emphasizes the complexity of coordinated virulence gene expression.

Although our data did not indicate a direct regulation of QS by either the TTSS or Act, we provide the first evidence for a positive correlation of these factors with QS, as their absence greatly reduced lactone production at high cell density, compared to the same conditions in the WT bacterium. Our future studies will focus on identifying the specific genes/regulators that are affected in these mutants.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the NIH/NIAID (AI41611) and the American Water Works Association Research Foundation. L. Pillai, a predoctoral fellow, obtained funding from the NIH T32 training grant in Emerging and Tropical Infectious Diseases. C. L. Galindo, a predoctoral fellow, obtained funding from the NSF. A. A. Fadl was supported by the McLaughlin Postdoctoral Fellowship.

We thank Mardelle Susman for editing the manuscript and Alfredo Torres for stimulating discussion.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, 301 University Blvd., UTMB, Galveston, TX 77555-1070. Phone: (409) 747-0578. Fax: (409) 747-6869. E-mail: achopra{at}utmb.edu. Back

Editor: J. T. Barbieri

{dagger} Equally contributed to the manuscript. Back


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Infection and Immunity, October 2005, p. 6446-6457, Vol. 73, No. 10
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