SUMMARY
Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of
invasive disease among children and the immunocompromised. While vaccine development efforts are ongoing,
the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect
high-risk individuals from infection. STm invades the intestinal mucosa before disseminating systemically.
Invasion requires injection of specific effector proteins into host cells through a Type Three Secretion System
(T3SS). Most of the structural components of the invasion-associated T3SS and its secreted effector proteins
are encoded in a genomic region known as Salmonella Pathogenicity Island 1 (SPI-1). Regulation of SPI-1 genes
represents a model system for how pathogenic bacteria respond to environmental signals to induce expression
of virulence genes. The master regulator of SPI-1 gene transcription is a DNA-binding transcription factor, HilD,
which is itself encoded within SPI-1. Five of the HilD-activated genes encode regulators, HilC, RtsA, InvF, SprB
and HilA, which we postulate are involved in temporal regulation of SPI-1 gene expression. However, very little
is known about how the different regulators contribute to the timing of expression of target genes during infection.
We comprehensively mapped the regulatory targets of HilD, HilC, RtsA, InvF, SprB and HilA, defining the
invasion “super-regulon”. Remarkably, the large majority of the >100 direct regulatory interactions we identified
were novel. By analyzing published data, we identified 12 members of the invasion super-regulon whose in vitro
expression profiles correlate with those of known invasion genes. We refer to these as “Invasion-Co-Regulated
Genes” (ICGs). A large-scale transposon mutagenesis study performed by another group suggests that most or
all of the ICGs are required for efficient infection of multiple animal models. We will dissect the function of ICGs
at different stages of infection using in vitro and in vivo infection models, and we will determine the expression
profiles of invasion super-regulon genes upon activation and inactivation in liquid growth and during infection of
epithelial cells in vitro, thereby defining the relationship between regulator and expression timing. The work
proposed here will represent the first step in establishing the role of uncharacterized genes that have strong ties
to the invasion process. We also expect to show that expression timing for invasion super-regulon genes is
determined by the associated transcription factors, which would represent an important advance in our
understanding of how regulatory networks contribute to bacterial pathogenesis.
