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Infection and Immunity, September 2002, p. 5319-5321, Vol. 70, No. 9
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.9.5319-5321.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of Genes Affecting Salmonella enterica Serovar Enteritidis Infection of Chicken Macrophages

Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL Utrecht, The Netherlands

Received 8 November 2001/ Returned for modification 5 February 2002/ Accepted 11 June 2002

	ABSTRACT

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Screening of 7,680 Salmonella enterica serovar Enteritidis mutants for attenuation in a chicken macrophage infection model yielded a series of mutants including several with defects in previously unrecognized Salmonella virulence genes. One of the newly identified genes was the pbpA2 gene, belonging to the penicillin binding protein gene family.

	TEXT

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Salmonella strains pose a major problem in public health, causing diseases ranging from gastroenteritis to typhoid fever. In recent years, Salmonella enterica serovar Enteritidis has replaced serovar Typhimurium as the primary etiologic agent of Salmonella infections in many countries (15). A likely source of serovar Enteritidis is the consumption of infected poultry (6, 15). The molecular mechanisms that enable this serovar to persist in poultry are poorly defined. To address this topic, we generated a mini-Tn10 mutant library of serovar Enteritidis strain CVI-1 and searched for cellular-infection-impaired mutants in a chicken macrophage infection model.

The mutant library was constructed by transforming strain CVI-1 with plasmid pKO3 carrying the mini-Tn10 delivery system from plasmid pLOF/KM (7). Induction of the transposition event eventually yielded 7,680 kanamycin-resistant colonies that were individually collected and tested for their ability to invade or survive in the chicken macrophage HD-11 cell line (5). After several rounds of selection which involved plate counting of the number of intracellular bacteria recovered after 30 min of infection and 2 h of treatment with colistin (100 µg/ml) to kill the extracellular microorganisms, 37 clones (designated SEM1 to SEM37) were identified that consistently yielded reduced (5 to 50% of wild-type) levels of cellular infection.

Genetic characterization of the selected mutants with inverse PCR using outward-oriented transposon primers followed by DNA sequencing of the products revealed the transposon insertion sites for 36 of the 37 mutants (Table 1). Sequence analysis indicated that in 14 strains (38%, group I in Table 1) genes had been disrupted that shared homology with genes involved in the assembly of flagella or bacterial motility in serovar Typhimurium. Analysis of the 37 selected mutants for their behavior in U-shaped tubes containing 0.4% Luria-Bertani soft agar demonstrated that all mutants in group I indeed were nonmotile. Bacterial motility has previously been demonstrated to expedite Salmonella entry into cultured epithelial cells, probably by enabling more rapid contact with the monolayer (13, 21). Fluorescence-activated cell sorting analysis with the serovar Enteritidis strain CVI-1 and the mutant strains made fluorescent by introduction of the plasmid pEGFP, encoding the green fluorescent protein, yielded similar results for chicken macrophages (data not shown), explaining the organisms' poor recovery from the cells.

Several of the metabolic genes defective in our mutants have not been previously identified as Salmonella virulence determinants. Apart from the PhoP/PhoQ-regulated ugd (pagA) gene, which in serovar Typhimurium has been demonstrated to be transcriptionally active inside macrophages and to be necessary for growth in a low-magnesium environment (1, 19, 20), two genes were identified with homology to the E. coli tdh gene and the cat2 gene in Clostridium aminobutyricum. These genes encode a threonine dehydrogenase (3) and a 4-hydroxybutyrate coenzyme A transferase (16), respectively. In E. coli, the tdh homologue is regulated by the lrp gene (9), which is one of the regulators of virulence genes in serovar Typhimurium (14). The function of the tdh gene in Salmonella has never been established. An additional previously unrecognized gene important for cellular infection of chicken macrophages was the serovar Enteritidis icd gene, which may encode the enzyme isocitrate dehydrogenase. This gene is considered to have a housekeeping function and appears to be conserved among most Salmonella serovars (22). Together, these results strongly suggest that metabolic adaptation is an important feature in serovar Enteritidis infection of chicken macrophages. Whether the newly identified genes also contribute to virulence of other Salmonella serovars and/or in other hosts awaits further study.

Another gene not previously implicated in Salmonella virulence was the pbpA2 gene defective in mutant SEM35. This gene exhibited approximately 60% homology with E. coli pbpA encoding penicillin binding protein 2 (4). Analysis of the serovar Typhimurium genome sequence indicated that this serovar carries two related pbpA sequences, one (designated pbpA) positioned adjacent to the putative rodA locus, and the other (designated pbpA2) flanking the argS locus. The latter gene was >99% identical in sequence to the gene disrupted in SEM35. To ascertain that the mutant phenotype of SEM35 was caused by disruption of pbpA2, intact copies of both the serovar Enteritidis pbpA and pbpA2 genes were PCR amplified, cloned onto plasmid pWSK29, and introduced into SEM35. Infection assays with HD-11 cells demonstrated that the intact pbpA2 gene completely restored the wild-type phenotype, while the pbpA gene was unable to complement the defect in SEM35 (Fig. 1). In serovar Typhimurium, the promoter of the pbpA2 gene is acid inducible (20), which could point to a role in intracellular survival. Furthermore, the homology of PbpA2 with the members of the penicillin binding protein family may indicate a role in cell wall synthesis and in the resistance against antimicrobial peptides (17). Thus, PbpA2 may contribute to intracellular survival of Salmonella by providing resistance to antimicrobial peptides in a low-pH environment. The finding that the pbpA gene was unable to complement pbpA2 function indicates that these two related genes have different functional properties.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Recovery of strains CVI-1 (positive control), Se857fliC mutant (nonflagellated, negative control) (21), SEM35 (pbpA2 mutant), and SEM35 carrying pWSK29::pbpA2 or SEM35 carrying pWSK29::pbpA, from the intracellular compartment of HD-11 macrophages after 30 min of infection and 2 h of colistin treatment. Data are the means ± standard deviations of three experiments.

	ACKNOWLEDGMENTS

 
K. N. Timmis is gratefully acknowledged for providing plasmid mini-Tn10-pLOF/KM.

This work was supported in part by grants from the CVVM and TNO, The Netherlands.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands. Phone: 31 30 253 4344. Fax: 31 30 254 0784. E-mail: j.vanputten{at}vet.uu.nl.

Editor: V. J. DiRita

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, Y. Articles by van Putten, J. P. M. Search for Related Content PubMed PubMed Citation Articles by Zhao, Y. Articles by van Putten, J. P. M.