Gut immune maturation depends on colonization with a host-specific microbiota.

Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.

The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis.

Bacterial pathogens sense host cues to activate expression of virulence genes. Most of these signals are sensed through histidine kinases (HKs), which comprise the main sensory mechanism in bacteria. The host stress hormones epinephrine (Epi) and norepinephrine are sensed through the QseC HK, which initiates a complex signaling cascade to regulate virulence gene expression in enterohemorrhagic Escherichia coli (EHEC). Epi signaling through QseC activates expression of the genes encoding the QseEF 2-component system. QseE is an HK, and QseF is a response regulator. Here, we show that QseE is a second bacterial adrenergic receptor that gauges the stress signals Epi, sulfate, and phosphate. The qseEF genes are organized within an unusual operonic structure, in that a gene is encoded between qseE and qseF. This gene was renamed qseG, and it was shown to encode an outer membrane (OM) protein. EHEC uses a type III secretion system (TTSS) to translocate effector proteins to the epithelial cells that rearrange the host cytoskeleton to form pedestal-like structures that cup the bacterium. QseE, QseG, and QseF are necessary for pedestal formation. Although QseE and QseF are involved in the transcriptional control of genes necessary for pedestal formation, QseG is necessary for translocation of effectors into epithelial cells. QseG is an OM protein necessary for translocation of TTSS effectors that also works in conjunction with a 2-component signaling system that senses host stress signals.

A transcriptome study of the QseEF two-component system and the QseG membrane protein in enterohaemorrhagic Escherichia coli O157 : H7.

QseE is a sensor kinase that responds to epinephrine, sulfate and phosphate. QseE constitutes a two-component signalling system together with the QseF sigma(54)-dependent response regulator. Encoded within the same operon as qseEF is the qseG gene, which encodes a membrane protein involved in the translocation of a type III secretion effector protein of enterohaemorrhagic Escherichia coli (EHEC) into epithelial cells. The qseEGF genes also form an operon with the glnB gene, which encodes the E. coli nitrogen sensor PII protein. Here we report a transcriptome analysis comparing qseE, qseF andqseG single mutants with the wild-type strain. This study revealed that the proteins encoded by these genes play a modest but significant role in iron uptake. Although QseEFG regulate genes involved in nitrogen utilization, these proteins do not play a notable role in nitrogen metabolism. In addition, QseEFG regulate transcription of the rcsBC and phoPQ two-component systems, linking several signal transduction pathways. The similarity of the microarray profiles of these mutants also indicates that these proteins work together. These data indicate that QseEFG are involved in the regulation of virulence and metabolism in EHEC.

Quorum sensing: the many languages of bacteria.

In the conventional view of prokaryotic existence, bacteria live unicellularly, with responses to external stimuli limited to the detection of chemical and physical signals of environmental origin. This view of bacteriology is now recognized to be overly simplistic, because bacteria communicate with each other through small 'hormone-like' organic compounds referred to as autoinducers. These bacterial cell-to-cell signaling systems were initially described as mechanisms through which bacteria regulate gene expression via cell density and, therefore, they have been collectively termed quorum sensing. The functions controlled by quorum sensing are varied and reflect the needs of a particular species of bacteria to inhabit a given niche. Three major quorum-sensing circuits have been described: one used primarily by Gram-negative bacteria, one used primarily by Gram-positive bacteria, and one that has been proposed to be universal.

In vivo imaging and tracking of host-microbiota interactions via metabolic labeling of gut anaerobic bacteria.

The intestine is densely populated by anaerobic commensal bacteria. These microorganisms shape immune system development, but understanding of host-commensal interactions is hampered by a lack of tools for studying the anaerobic intestinal environment. We applied metabolic oligosaccharide engineering and bioorthogonal click chemistry to label various commensal anaerobes, including Bacteroides fragilis, a common and immunologically important commensal. We studied the dissemination of B. fragilis after acute peritonitis and characterized the interactions of the intact microbe and its polysaccharide components in myeloid and B cell lineages. We were able to assess the distribution and colonization of labeled B. fragilis along the intestine, as well as niche competition after coadministration of multiple species of the microbiota. We also fluorescently labeled nine additional commensals (eight anaerobic and one microaerophilic) from three phyla common in the gut--Bacteroidetes, Firmicutes and Proteobacteria--as well as one aerobic pathogen (Staphylococcus aureus). This strategy permits visualization of the anaerobic microbial niche by various methods, including intravital two-photon microscopy and non-invasive whole-body imaging, and can be used to study microbial colonization and host-microbe interactions in real time.

The starting lineup: key microbial players in intestinal immunity and homeostasis.

The complexity of microbiota inhabiting the intestine is increasingly apparent. Delicate balance of numerous bacterial species can affect development of the immune system, how susceptible a host is to pathogenic organisms, and the auto-inflammatory state of the host. In the last decade, with the increased use of germ-free mice, gnotobiotic mice, and animal models in which a germ-free animal has been colonized with a foreign microbiota such as humanized mice, it has been possible to delineate relationships that specific bacteria have with the host immune system and to show what role they may play in overall host health. These models have not only allowed us to tease out the roles of individual species, but have also allowed the discovery and characterization of functionally unknown organisms. For example, segmented filamentous bacteria (SFB) have been shown to play a vital role in expansion of IL-17 producing cells. Prior to linking their key role in immune system development, little was known about these organisms. Bacteroides fragilis can rescue some of the immune defects of gnotobiotic mice after mono-colonization and have anti-inflammatory properties that can alleviate colitis and experimental allergic encephalitis in murine models. Additionally, Clostridium species have most recently been shown to expand regulatory T-cell populations leading to anti-inflammatory conditions. This review will highlight and summarize some of the major findings within the last decade concerning the role of select groups of bacteria including SFB, Clostridium, Bacteroides, Bifidobacterium, and Lactobacillus, and their impact on host mucosal immune systems.

Targeting QseC signaling and virulence for antibiotic development.

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.

A novel two-component signaling system that activates transcription of an enterohemorrhagic Escherichia coli effector involved in remodeling of host actin.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for worldwide outbreaks of bloody diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. After colonizing the large intestine, EHEC forms attaching and effacing (AE) lesions on intestinal epithelial cells. These lesions cause destruction of the microvilli and elicit actin rearrangement to form pedestals that cup each bacterium individually. EHEC responds to a signal produced by the intestinal microbial flora, autoinducer-3 (AI-3), and the host hormones epinephrine and norepinephrine to activate transcription of the genes involved in AE lesion formation. These three signals, involved in interkingdom communication, are sensed by bacterial sensor kinases. Here we describe a novel two-component system, QseEF (quorum-sensing E. coli regulators E and F), which is part of the AI-3/epinephrine/norepinephrine signaling system. QseE is the sensor kinase and QseF the response regulator. The qseEF genes are cotranscribed, and transcription of qseEF is activated by epinephrine through the QseC sensor. A qseF mutant does not form AE lesions. QseF activates transcription of the gene encoding EspFu, an effector protein translocated to the host cell by the EHEC, which mimics a eukaryotic SH2/SH3 adapter protein to engender actin polymerization during pedestal formation. Expression of the espFu gene from a plasmid restored AE lesion formation to the qseF mutant, suggesting that lack of espFu expression in this mutant was responsible for the loss of pedestal formation. These findings suggest the QseEF is a two-component system involved in the regulation of AE lesion formation by EHEC.