Enteropathogenic Escherichia coli and enterohemorrhagic E. coli are diarrheagenic bacterial human pathogens that cause severe gastroenteritis. These enteric pathotypes, together with the mouse pathogen Citrobacter rodentium, belong to the family of attaching and effacing pathogens that form a distinctive histological lesion in the intestinal epithelium. The virulence of these bacteria depends on a type III secretion system (T3SS), which mediates the translocation of effector proteins from the bacterial cytosol into the infected cells. The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial membranes, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. In addition, two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows these pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform. In this review, we present recent advances in the understanding of the T3SS in attaching and effacing pathogens.

Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.

Enteropathogenic (EPEC) and enterohemorrhagic (EHEC)Escherichia coliare clinically important diarrheagenic pathogens that adhere to the intestinal epithelial surface. TheE. colicommon pili (ECP), or meningitis-associated and temperature-regulated (MAT) fimbriae, are ubiquitous among both commensal and pathogenicE. colistrains and play a role as colonization factors by promoting the interaction between bacteria and host epithelial cells and favoring interbacterial interactions in biofilm communities. The first gene of theecpoperon encodes EcpR (also known as MatA), a proposed regulatory protein containing a LuxR-like C-terminal helix-turn-helix (HTH) DNA-binding motif. In this work, we analyzed the transcriptional regulation of theecpgenes and the role of EcpR as a transcriptional regulator. EHEC and EPECecpRmutants produce less ECP, while plasmids expressing EcpR increase considerably the expression of EcpA and production of ECP. Theecpgenes are transcribed as an operon from a promoter located 121 bp upstream of the start codon ofecpR. EcpR positively regulates this promoter by binding to two TTCCT boxes distantly located upstream of theecppromoter, thus enhancing expression of downstreamecpgenes, leading to ECP production. EcpR mutants in the putative HTH DNA-binding domain are no longer able to activateecpexpression or bind to the TTCCT boxes. EcpR-mediated activation is aided by integration host factor (IHF), which is essential for counteracting the repression exerted by histone-like nucleoid-structuring protein (H-NS) on theecppromoter. This work demonstrates evidence about the interplay between a novel member of a diverse family of regulatory proteins and global regulators in the regulation of a fimbrial operon.

Background: In recent years Staphylococcus epidermidis has been considered an important and frequent causative agent of health care-associated infections (HAIs), increasing the costs of hospitalization, morbidity, and mortality. Antibiotic resistance and biofilm formation are the most important obstacles in the treatment of infections caused by this microorganism. The aim of this work was to determine the most prevalent STs, as well as the antibiotic resistance profile and biofilm formation of S. epidermidis clinical isolates obtained from hospitalized patients in two hospitals in Acapulco, Guerrero in two time periods. Methods: Twenty methicillin-resistant S. epidermidis strains isolated from patients with bacteremia in two hospitals in two time periods were analyzed. Identification and antibiotic susceptibility were performed using the Vitek automated system. Molecular confirmation of the identification and methicillin resistance was performed by duplex PCR of the mecA and nuc genes. Biofilm production was analyzed, and the clonal origin was determined by multilocus sequence typing (MLST). Results: We identified 14 antibiotic resistance profiles as well as 13 sequence types (ST), including the new ST761. We also found that ST2 and ST23 were the most prevalent and, together with ST59, were found in both time periods. Seventeen of our clinical isolates were multidrug-resistant, but all of them were sensitive to linezolid and vancomycin, and this was not related to biofilm production. Additionally, we standardized a duplex PCR to identify methicillin-resistant S. epidermidis strains. In conclusion, S. epidermidis STs 2, 23, and 59 were found in both time periods. This study is the first report of S. epidermidis ST761. The clinical isolates obtained in this work showed a high multidrug resistance that is apparently not related to biofilm production.

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Enteropathogenic Escherichia coli uses a type III secretion system (T3SS), encoded in the locus of enterocyte effacement (LEE) pathogenicity island, to translocate a wide repertoire of effector proteins into the host cell in order to subvert cell signaling cascades and promote bacterial colonization and survival. Genes encoding type III-secreted effectors are located in the LEE and scattered throughout the chromosome. While LEE gene regulation is better understood, the conditions and factors involved in the expression of effectors encoded outside the LEE are just starting to be elucidated. Here, we identified a highly conserved sequence containing a 13-bp inverted repeat (IR), located upstream of a subset of genes coding for different non-LEE-encoded effectors in A/E pathogens. Site-directed mutagenesis and deletion analysis of the nleH1 and nleB2 regulatory regions revealed that this IR is essential for the transcriptional activation of both genes. Growth conditions that favor the expression of LEE genes also facilitate the activation of nleH1 and nleB2; however, their expression is independent of the LEE-encoded positive regulators Ler and GrlA but is repressed by GrlR and the global regulator H-NS. In contrast, GrlA and Ler are required for nleA expression, while H-NS silences it. Consistent with their role in the regulation of nleA, purified Ler and H-NS bound to the regulatory region of nleA upstream of its promoter. This work shows that at least two modes of regulation control the expression of effector genes in attaching and effacing (A/E) pathogens, suggesting that a subset of effector functions may be coordinately expressed in a particular niche or time during infection.