Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.

Previous studies have shown that Listeria monocytogenes flagellar motility genes, including flaA, encoding flagellin, are transcriptionally down-regulated at 37°C. For some L. monocytogenes strains, temperature-dependent motility gene expression is less stringent. By using flaA-lacZ transcriptional fusions, we identified regions upstream of the ؊35͞؊10 promoter elements that are necessary for temperature-dependent expression of flaA in L. monocytogenes strain EGDe. Whereas the sequence of the flaA promoter region was identical in L. monocytogenes strain 10403S, transcriptional activity was only partially down-regulated at 37°C in 10403S. This finding suggested that a transacting regulatory protein with differential expression or activity in EGDe might be involved in temperature-dependent transcription of flaA. Indeed, a protein factor capable of specifically binding to the flaA promoter region was identified in cytoplasmic extracts of EGDe by using affinity purification and MS. Deletion of the factor-encoding gene (lmo0674) resulted in loss of temperature-dependent flaA expression and an increase in flaA promoter activity. Expression of other motility genes was also deregulated in the lmo0674 deletion. We have designated lmo0674 as mogR, indicating its role as a motility gene repressor. In tissue culture models, MogR repression of flaA during intracellular infection was independent of temperature and a deletion of mogR reduced the capacity for cell-to-cell spread. During in vivo infection, a deletion of mogR resulted in a 250-fold decrease in virulence. These studies indicate that regulation of flagellar motility gene expression and͞or other genes controlled by MogR is required for full virulence of L. monocytogenes. Listeria monocytogenes is a food-borne bacterial pathogen of humans and is best known for its intricate intracellular lifestyle (1). The majority of genes encoding virulence factors required for intracellular infection, such as ActA, which is necessary for actin-based motility and cell-to-cell spread (2, 3), are coordinately regulated by the transcriptional activator protein PrfA (4). L. monocytogenes can also swim by means of flagella-based motility in extracellular environments. Previous studies have shown that flagellar motility gene expression in L. monocytogenes is regulated by temperature. L. monocytogenes strains are highly flagellated and motile at low temperatures, 30°C and below, and are typically not motile at temperatures of 37°C or above (5, 6). Furthermore, bacterial flagellins serve as pattern recognition molecules for Toll-like receptor 5-mediated signaling, leading to activation of innate immune responses to infection (7,8). Because previous studies (9, 10) have shown that transcription of L. monocytogenes flaA, encoding flagellin, is down-regulated at physiological temperature (37°C), it has been proposed that down regulation of flaA expression during in vivo infection by L. monocytogenes may serve as an adaptive mechanism to avoid host recognition and mobilization of host innate i...

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

Neocentromeres are ectopic sites where new functional kinetochores assemble and permit chromosome segregation. Neocentromeres usually form following genomic alterations that remove or disrupt centromere function. The ability to form neocentromeres is conserved in eukaryotes ranging from fungi to mammals. Neocentromeres that rescue chromosome fragments in cells with gross chromosomal rearrangements are found in several types of human cancers, and in patients with developmental disabilities. In this review, we discuss the importance of neocentromeres to human health and evaluate recently developed model systems to study neocentromere formation, maintenance, and function in chromosome segregation. Additionally, studies of neocentromeres provide insight into native centromeres; analysis of neocentromeres found in human clinical samples and induced in model organisms distinguishes features of centromeres that are dependent on centromere DNA from features that are epigenetically inherited together with the formation of a functional kinetochore.