A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.Listeria monocytogenes is a gram-positive, food-borne bacterium that is widely distributed in nature and can cause serious infection in susceptible individuals (9). The infection pathogenesis of this facultative intracellular pathogen includes phases where the bacteria successfully proliferate in the challenging environment in a variety of mammalian cell types like epithelial cells, endothelial cells, hepatocytes, dendritic cells, and macrophages. While invading the host cells, L. monocytogenes is confronted with suboptimal growth conditions, such as a rapid pH drop within the phagosome (1). Following early escape from the primary phagosome by membrane lysis, the pathogen is released into the cytosol where it encounters conditions of nutrient and iron starvation. The genes essential for the internalization of L. monocytogenes by the mammalian host cells (inlA and inlB), phagosomal escape (hly, plcA, and plcB), and intra-and intercellular motility (actA) have been extensively studied in the past years (45). All these virulence genes are controlled by PrfA, a central transcription regulator, which activates gene transcription by binding to a specific site termed the PrfA box and recruiting RNA polymerase to the PrfAdependent promot...

Host recognition of microbial components is essential in mediating an effective immune response. Cytosolic bacteria must secure entry into the host cytoplasm to facilitate replication, and in doing so, liberating microbial ligands which activate cytosolic innate immune sensors and the inflammasome. Here, we identified a multi-component enterotoxin hemolysin BL (HBL) which

Intracellular bacterial pathogens (IBPs) are dependent on various nutrients provided by the host cells. Different strategies may therefore be necessary to adapt the intracellular metabolism of IBPs to the host cells. The specific carbon sources, the catabolic pathways participating in their degradation, and the biosynthetic performances of IBPs are still poorly understood. In this report, we have exploited the technique of 13C-isotopologue profiling to further study the carbon metabolism of Listeria monocytogenes by using the EGDe wild-type strain and mutants (defective in the uptake and/or catabolism of various carbon compounds) replicating in J774A.1 macrophages. For this goal, the infected macrophages were cultivated in the presence of [1,2-13C2]glucose, [U-13C3]glycerol, [U-13C3]pyruvate, [U-13C3]lactate, or a mix of [U-13C]amino acids. GC/MS-based isotopologue profiling showed efficient utilization of amino acids, glucose 6-phosphate, glycerol, and (at a low extent) also of lactate but not of pyruvate by the IBPs. Most amino acids imported from the host cells were directly used for bacterial protein biosynthesis and hardly catabolized. However, Asp was de novo synthesized by the IBPs and not imported from the host cell. As expected, glycerol was catabolized via the ATP-generating lower part of the glycolytic pathway, but apparently not used for gluconeogenesis. The intermediates generated from glucose 6-phosphate in the upper part of the glycolytic pathway and the pentose phosphate shunt likely serve primarily for anabolic purposes (probably for the biosynthesis of cell wall components and nucleotides). This bipartite bacterial metabolism which involves at least two major carbon substrates—glycerol mainly for energy supply and glucose 6-phosphate mainly for indispensible anabolic performances—may put less nutritional stress on the infected host cells, thereby extending the lifespan of the host cells to the benefit of the IBPs.

Multidrug resistant microorganisms are forecast to become the single biggest challenge to medical care in the 21st century. Over the last decades, members of the genus Acinetobacter have emerged as bacterial opportunistic pathogens, in particular as challenging nosocomial pathogens because of the rapid evolution of antimicrobial resistances. Although we lack fundamental biological insight into virulence mechanisms, an increasing number of researchers are working to identify virulence factors and to study antibiotic resistance. Here, we review current knowledge regarding the regulation of virulence genes and antibiotic resistance in Acinetobacter baumannii. A survey of the two-component systems AdeRS, BaeSR, GacSA and PmrAB explains how each contributes to antibiotic resistance and virulence gene expression, while BfmRS regulates cell envelope structures important for pathogen persistence. A. baumannii uses the transcription factors Fur and Zur to sense iron or zinc depletion and upregulate genes for metal scavenging as a critical survival tool in an animal host. Quorum sensing, nucleoid-associated proteins, and non-classical transcription factors such as AtfA and small regulatory RNAs are discussed in the context of virulence and antibiotic resistance.