SummaryEnteroaggregative Escherichia coli (EAEC) is defined by aggregative adherence (AA) to HEp-2 cells, where bacteria display adherence to cell surfaces and also to the intervening substratum in a stacked-brick configuration. We previously showed that an AraC homologue designated AggR is required for the expression of plasmid-encoded genes that mediate AA of EAEC strain 042. In this study, we hypothesized that AggR also controls the expression of other virulence determinants in EAEC 042. Using proteomic and microarray analysis, we identified for the first time that AggR activates the expression of chromosomal genes, including 25 contiguous genes (aaiA-Y), which are localized to a 117 kb pathogenicity island (PAI) inserted at pheU. Many of these genes have homologues in other Gram-negative bacteria and were recently proposed to constitute a type VI secretion system (T6SS). AaiC was identified as a secreted protein that has no apparent homologues within GenBank. EAEC strains carrying in-frame deletions of aaiB, aaiG, aaiO or aaiP still synthesized AaiC; however, AaiC secretion was abolished. Cloning of aai genes into E. coli HB101 suggested that aaiA-P are sufficient for AaiC secretion. A second T6SS was identified within the pheU PAI that secretes a protein unrelated by sequence identity to AaiC. Distribution studies indicated that aaiA and aaiC are commonly found in EAEC isolates worldwide, particularly in strains defined as typical EAEC. These data support the hypothesis that AggR is a global regulator of EAEC virulence determinants, and builds on the hypothesis that T6SS is an importance mediator of pathogenesis.

AggR is a transcriptional regulator of enteroaggregative Escherichia coli (EAEC) and has been proposed as the defining factor for typical EAEC strains. Expression of multiple putative virulence factors, including the aggregative adherence fimbriae (AAF), dispersin, the dispersin translocator Aat, and the Aai type VI secretion system, have been found to be regulated by AggR. Here, we confirm the existence of at least 44 AggR-regulated genes using DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR); these genes include chromosomal and plasmid-borne loci and 19 previously unsuspected genes. Two previously uncharacterized virulence plasmid-encoded open reading frames (ORFs) (designated ORF3 and ORF4) exhibit significant identity with isoprenoid biosynthesis genes of Bacteria and Archaea. The predicted ORF4 product is closely related to isopentenyl isomerase (IDI) enzymes, whereas the predicted product of the adjacent ORF3 exhibits an aspartate-rich region that is common among trans-isoprenyl phosphate synthases. We show that mutations in these ORFs confer changes in bacterial surface properties. AggR coordinately controls expression of a large number of EAEC genes.

The primary function of the respiratory system of gas exchange renders it vulnerable to environmental pathogens that circulate in the air. Physical and cellular barriers of the respiratory tract mucosal surface utilize a variety of strategies to obstruct microbe entry. Physical barrier defenses including the surface fluid replete with antimicrobials, neutralizing immunoglobulins, mucus, and the epithelial cell layer with rapidly beating cilia form a near impenetrable wall that separates the external environment from the internal soft tissue of the host. Resident leukocytes, primarily of the innate immune branch, also maintain airway integrity by constant surveillance and the maintenance of homeostasis through the release of cytokines and growth factors. Unfortunately, pathogens such as influenza virus and Streptococcus pneumoniae require hosts for their replication and dissemination, and prey on the respiratory tract as an ideal environment causing severe damage to the host during their invasion. In this review, we outline the host-pathogen interactions during influenza and post-influenza bacterial pneumonia with a focus on interand intra-cellular crosstalk important in pulmonary immune responses.

Enteroaggregative Escherichia coli (EAEC) causes diarrhea in diverse populations worldwide. The AraC-like regulator AggR is a key virulence regulator in EAEC. AggR-regulated genes include those encoding the Aggregative Adherence Fimbria, the dispersin protein, and a type VI secretion system. This study characterizes the regulation of the aggR promoter (P(aggR)). Using primer extension analysis, the transcriptional start site of the aggR promoter was located 40 nucleotides upstream of the translational start. P(aggR) was found to be autoregulated and DNA footprinting revealed the presence of two AggR-binding sites: one upstream of the transcriptional start site and one downstream. Additionally, P(aggR) was found to be positively regulated by the DNA-binding protein FIS and negatively regulated by the global regulator H-NS. To further understand this complex regulation scheme, a bacterial luciferase reporter system was used with a mouse model of EAEC colonization. This allowed for the in vivo measurement of P(aggR), P(fis), and P(hns) activity. EAEC present in the mouse intestine possessed relatively high levels of P(fis) and P(aggR) activity and a low level of P(hns) when compared with in vitro experiments. The data provide significant insights into the regulation cascade leading to aggR expression in the mammalian intestine during EAEC infection.

Enteroaggregative Escherichia coli (EAEC) causes diarrhea in diverse populations worldwide. The AraC-like regulator AggR is a key virulence regulator in EAEC. AggR-regulated genes include those encoding the Aggregative Adherence Fimbria, the dispersin protein, and a type VI secretion system. This study characterizes the regulation of the aggR promoter (P(aggR)). Using primer extension analysis, the transcriptional start site of the aggR promoter was located 40 nucleotides upstream of the translational start. P(aggR) was found to be autoregulated and DNA footprinting revealed the presence of two AggR-binding sites: one upstream of the transcriptional start site and one downstream. Additionally, P(aggR) was found to be positively regulated by the DNA-binding protein FIS and negatively regulated by the global regulator H-NS. To further understand this complex regulation scheme, a bacterial luciferase reporter system was used with a mouse model of EAEC colonization. This allowed for the in vivo measurement of P(aggR), P(fis), and P(hns) activity. EAEC present in the mouse intestine possessed relatively high levels of P(fis) and P(aggR) activity and a low level of P(hns) when compared with in vitro experiments. The data provide significant insights into the regulation cascade leading to aggR expression in the mammalian intestine during EAEC infection.

There is substantial morbidity associated with severe respiratory infection due to human rhino/enteroviruses in children. Mortality was less severe than reported in other respiratory viruses such as influenza and respiratory syncytial virus. The burden of illness from human rhino/enteroviruses in the ICU in terms of resource utilization may be considerable.

Lactobacillus GG elaborates small peptides showing various degrees of antibacterial activity. NPSRQERR showed the most potent antibacterial effect that was detected both in Gram-negative and Gram-positive microorganisms. These synthetic peptides may represent novel tools for the treatment of bacterial infectious diseases.

Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.