The chaperone PrsA2 regulates the secretion, stability, and folding of listeriolysin O during infection.

Unlabelled: Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2.

The trophoblast surface becomes refractory to adhesion by congenitally transmitted and during cytotrophoblast to syncytiotrophoblast development.

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with .

secretion chaperones PrsA, SlrA, and HtrA are required for competence, antibiotic resistance, colonization, and invasive disease.

is a Gram-positive bacterium and a significant health threat with the populations most at risk being children, the elderly, and the immuno-compromised. To colonize and transition into an invasive infectious organism, secretes virulence factors that are translocated across the bacterial membrane and destined for surface exposure, attachment to the cell wall, or secretion into the host. The surface exposed protein chaperones PrsA, SlrA, and HtrA facilitate protein secretion; however, the distinct roles contributed by each of these secretion chaperones have not been well defined.

Listeria monocytogenes two component system PieRS regulates secretion chaperones PrsA1 and PrsA2 and enhances bacterial translocation across the intestine.

Listeria monocytogenes (Lm) is a widespread environmental Gram-positive bacterium that can transition into a pathogen following ingestion by a susceptible host. To cross host barriers and establish infection, Lm is dependent upon the regulated secretion and activity of many proteins including PrsA2, a peptidyl-prolyl cis-trans isomerase with foldase activity. PrsA2 contributes to the stability and activity of a number of secreted virulence factors that are required for Lm invasion, replication, and cell-to-cell spread within the infected host.

An mRNA-mRNA Interaction Couples Expression of a Virulence Factor and Its Chaperone in Listeria monocytogenes.

Bacterial pathogens often employ RNA regulatory elements located in the 5' untranslated regions (UTRs) to control gene expression. Using a comparative structural analysis, we examine the structure of 5' UTRs at a global scale in the pathogenic bacterium Listeria monocytogenes under different conditions. In addition to discovering an RNA thermoswitch and detecting simultaneous interaction of ribosomes and small RNAs with mRNA, we identify structural changes in the 5' UTR of an mRNA encoding the post-translocation chaperone PrsA2 during infection conditions.

Altering the Guanine Quadruplex Loop Bases Affects Pilin Antigenic Variation.

possesses a programmed recombination system that allows the bacteria to alter the major subunit of the type IV pilus, pilin or PilE. An alternate DNA structure known as a guanine quadruplex (G4) is required for pilin antigenic variation (pilin Av). The G-C base pairs within the G4 motif are required for pilin Av, but simple mutation of the loop bases does not affect pilin Av.

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av).

Transferase Versus Hydrolase: The Role of Conformational Flexibility in Reaction Specificity.

Active in the aqueous cellular environment where a massive excess of water is perpetually present, enzymes that catalyze the transfer of an electrophile to a non-water nucleophile (transferases) require specific strategies to inhibit mechanistically related hydrolysis reactions. To identify principles that confer transferase versus hydrolase reaction specificity, we exploited two enzymes that use highly similar catalytic apparatuses to catalyze the transglycosylation (a transferase reaction) or hydrolysis of α-1,3-glucan linkages in the cyclic tetrasaccharide cycloalternan (CA). We show that substrate binding to non-catalytic domains and a conformationally stable active site promote CA transglycosylation, whereas a distinct pattern of active site conformational change is associated with CA hydrolysis.

Structure to function of an α-glucan metabolic pathway that promotes Listeria monocytogenes pathogenesis.

Here we employ a 'systems structural biology' approach to functionally characterize an unconventional α-glucan metabolic pathway from the food-borne pathogen Listeria monocytogenes (Lm). Crystal structure determination coupled with basic biochemical and biophysical assays allowed for the identification of anabolic, transport, catabolic and regulatory portions of the cycloalternan pathway. These findings provide numerous insights into cycloalternan pathway function and reveal the mechanism of repressor, open reading frame, kinase (ROK) transcription regulators.

A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence.

Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth.

Identification of Conserved and Species-Specific Functions of the Listeria monocytogenes PrsA2 Secretion Chaperone.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis/trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence.

Listeria monocytogenes virulence factor secretion: don't leave the cell without a chaperone.

In Gram-positive bacteria, the secretion of proteins requires translocation of polypeptides across the bacterial membrane into the highly charged environment of the membrane-cell wall interface. Here, proteins must be folded and often further delivered across the matrix of the cell wall. While many aspects of protein secretion have been well studied in Gram-negative bacteria which possess both an inner and outer membrane, generally less attention has been given to the mechanics of protein secretion across the single cell membrane of Gram-positive bacteria.

Neisseria gonorrhoeae RecQ helicase HRDC domains are essential for efficient binding and unwinding of the pilE guanine quartet structure required for pilin antigenic variation.

The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE.

Transcription of a cis-acting, noncoding, small RNA is required for pilin antigenic variation in Neisseria gonorrhoeae.

The strict human pathogen Neisseria gonorrhoeae can utilize homologous recombination to generate antigenic variability in targets of immune surveillance. To evade the host immune response, N. gonorrhoeae promotes high frequency gene conversion events between many silent pilin copies and the expressed pilin locus (pilE), resulting in the production of variant pilin proteins.

RecA-binding pilE G4 sequence essential for pilin antigenic variation forms monomeric and 5' end-stacked dimeric parallel G-quadruplexes.

Neisseria gonorrhoeae is an obligate human pathogen that can escape immune surveillance through antigenic variation of surface structures such as pili. A G-quadruplex-forming (G4) sequence (5'-G(3)TG(3)TTG(3)TG(3)) located upstream of the N. gonorrhoeae pilin expression locus (pilE) is necessary for initiation of pilin antigenic variation, a recombination-based, high-frequency, diversity-generation system.

Focusing homologous recombination: pilin antigenic variation in the pathogenic Neisseria.

Some pathogenic microbes utilize homologous recombination to generate antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyse dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well-characterized pilin antigenic variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented.

The Neisseria gonorrhoeae photolyase orthologue phrB is required for proper DNA supercoiling but does not function in photo-reactivation.

Neisseria gonorrhoeae (Gc) is an obligate human pathogen and the causative agent of the sexually transmitted infection, gonorrhoea. Despite the fact that the gonococcus is not normally exposed to UV irradiation or visible light, the bacterium expresses a phrB orthologue, which in other organisms encodes a DNA photolyase that repairs UV-induced pyrimidine dimers with energy provided by visible light. We show that a Gc phrB mutant is not more sensitive to UV irradiation, independent of visible light exposure, and that the Gc phrB cannot complement an Escherichia coli phrB mutant strain.

An alternative DNA structure is necessary for pilin antigenic variation in Neisseria gonorrhoeae.

Pathogens can use DNA recombination to promote antigenic variation (Av) of surface structures to avoid immune detection. We identified a cis-acting DNA sequence near the antigenically variable pilin locus of the human pathogen, Neisseria gonorrhoeae. This 16-base pair guanine (G)-rich sequence was required for pilin Av and formed a guanine quartet (G4) structure in vitro.

Pathways change in expression during replicative aging in Saccharomyces cerevisiae.

Yeast replicative aging is a process resembling replicative aging in mammalian cells. During aging, wild-type haploid yeast cells enlarge, become sterile, and undergo nucleolar enlargement and fragmentation; we sought gene expression changes during the time of these phenotypic changes. Gene expression studied via microarrays and quantitative real-time reverse-transcription polymerase chain reaction (qPCR) has shown reproducible, statistically significant changes in messenger RNA (mRNA) of genes at 12 and 18-20 generations.

An inexpensive gel electrophoresis-based polymerase chain reaction method for quantifying mRNA levels.

In order to engage their students in a core methodology of the new genomics era, an ever-increasing number of faculty at primarily undergraduate institutions are gaining access to microarray technology. Their students are conducting successful microarray experiments designed to address a variety of interesting questions. A next step in these teaching and research laboratory projects is often validation of the microarray data for individual selected genes.