lacZ Reporter System as a Tool to Study Virulence Gene Regulation in Bacterial Pathogens

ABSTRACTTo adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes ofHaemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog ofhfq. Insertional inactivation ofhfqaltered the expression of ~16% of theH. ducreyigenes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in thehfqinactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation ofhfqdownregulated genes in theflp-tadandlspB-lspA2operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarkedhfqdeletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion ofhfqdownregulated Flp1 and impaired the ability ofH. ducreyito form microcolonies, downregulated DsrA and renderedH. ducreyiserum susceptible, and downregulated LspB and LspA2, which allowH. ducreyito resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor ofH. ducreyistationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog.IMPORTANCEPathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, includingHaemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required forH. ducreyito infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.

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Hfr mapping of mutations in Bordetella pertussis that define a genetic locus involved in virulence gene regulation.

Journal of Bacteriology ◽

10.1128/jb.176.23.7260-7266.1994 ◽

1994 ◽

Vol 176 (23) ◽

pp. 7260-7266 ◽

Cited By ~ 13

Author(s):

S Stibitz ◽

N H Carbonetti

Keyword(s):

Gene Regulation ◽

Bordetella Pertussis ◽

Genetic Locus ◽

Virulence Gene ◽

Virulence Gene Regulation

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Virulence Gene Regulation inShigella

EcoSal ◽

10.1128/ecosal.8.9.3 ◽

2004 ◽

Author(s):

Charles J. Dorman

Keyword(s):

Gene Regulation ◽

Virulence Gene ◽

Virulence Gene Regulation

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Virulence gene regulation inSalmonella enterica

Annals of Medicine ◽

10.3109/07853890109002075 ◽

2001 ◽

Vol 33 (3) ◽

pp. 178-185 ◽

Cited By ~ 26

Author(s):

Mark Clements ◽

Sofia Eriksson ◽

Dilek Tezcan-Merdol ◽

Jay C D Hinton ◽

Mikael Rhen

Keyword(s):

Gene Regulation ◽

Virulence Gene ◽

Virulence Gene Regulation

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Phasevarion-Regulated Virulence in the Emerging Pediatric Pathogen Kingella kingae

Infection and Immunity ◽

10.1128/iai.00319-17 ◽

2017 ◽

Vol 85 (12) ◽

Cited By ~ 9

Author(s):

Yogitha N. Srikhanta ◽

Ka Yee Fung ◽

Georgina L. Pollock ◽

Vicki Bennett-Wood ◽

Benjamin P. Howden ◽

...

Keyword(s):

Gene Regulation ◽

Heat Shock ◽

Virulence Gene ◽

Dna Methyltransferases ◽

Epigenetic Mechanism ◽

Phase Variable ◽

Kingella Kingae ◽

Content Type ◽

Associated Proteins ◽

Virulence Gene Regulation

ABSTRACT Kingella kingae is a common etiological agent of pediatric osteoarticular infections. While current research has expanded our understanding of K. kingae pathogenesis, there is a paucity of knowledge about host-pathogen interactions and virulence gene regulation. Many host-adapted bacterial pathogens contain phase variable DNA methyltransferases (mod genes), which can control expression of a regulon of genes (phasevarion) through differential methylation of the genome. Here, we identify a phase variable type III mod gene in K. kingae, suggesting that phasevarions operate in this pathogen. Phylogenetic studies revealed that there are two active modK alleles in K. kingae. Proteomic analysis of secreted and surface-associated proteins, quantitative PCR, and a heat shock assay comparing the wild-type modK1 ON (i.e., in frame for expression) strain to a modK1 OFF (i.e., out of frame) strain revealed three virulence-associated genes under ModK1 control. These include the K. kingae toxin rtxA and the heat shock genes groEL and dnaK. Cytokine expression analysis showed that the interleukin-8 (IL-8), IL-1β, and tumor necrosis factor responses of THP-1 macrophages were lower in the modK1 ON strain than in the modK1::kan mutant. This suggests that the ModK1 phasevarion influences the host inflammatory response and provides the first evidence of this phase variable epigenetic mechanism of gene regulation in K. kingae.

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