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Enteric pathogens such as enterohemorrhagic E. coli (EHEC) and its surrogate murine model Citrobacter rodentium sense indole levels within the gut to navigate its biogeography and modulate virulence gene expression. Indole is a microbiota-derived signal that is more abundant in the intestinal lumen, with its concentration decreasing at the epithelial lining where it is absorbed. E. coli, but not C. rodentium, produces endogenous indole because it harbors the

The interactions between the gut microbiota and pathogens are complex and can determine the outcome of an infection. Enterohemorrhagic Escherichia coli (EHEC) is a major human enteric pathogen that colonizes the colon through attaching and effacing (AE) lesions and uses microbiota-derived molecules as cues to control its virulence. Different gut commensals can modulate EHEC virulence. However, the lack of an animal model that recapitulates the human pathophysiology of EHEC infection makes it challenging to investigate how variations in microbiota composition could affect host susceptibility to this pathogen. Here, we addressed these interactions building from simple to more complex

Polyunsaturated fatty acids (PUFAs) play important roles in host immunity. Manipulation of lipid content in host tissues through diet or pharmacological interventions is associated with altered severity of various inflammatory diseases., The mammalian gastrointestinal tract is a complex biochemical organ that generates a diverse milieu of host- and microbe-derived metabolites. In this environment, bacterial pathogens sense and respond to specific stimuli, which are integrated into the regulation of their virulence programs. Previously, we identified the transcription factor FadR, a long-chain fatty acid (LCFA) acyl coenzyme A (acyl-CoA) sensor, as a novel virulence regulator in the human foodborne pathogen enterohemorrhagic Escherichia coli (EHEC). Here, we demonstrate that exogenous LCFAs directly inhibit the locus of enterocyte effacement (LEE) pathogenicity island in EHEC through sensing by FadR. Moreover, in addition to LCFAs that are 18 carbons in length or shorter, we introduce host-derived arachidonic acid (C20:4) as an additional LCFA that is recognized by the FadR system in EHEC. We show that arachidonic acid is processed by the acyl-CoA synthetase FadD, which permits binding to FadR and decreases FadR affinity for its target DNA sequences. This interaction enables the transcriptional regulation of FadR-responsive operons by arachidonic acid in EHEC, including the LEE. Finally, we show that arachidonic acid inhibits hallmarks of EHEC disease in a FadR-dependent manner, including EHEC attachment to epithelial cells and the formation of attaching and effacing lesions. Together, our findings delineate a molecular mechanism demonstrating how LCFAs can directly inhibit the virulence of an enteric bacterial pathogen. More broadly, our findings expand the repertoire of ligands sensed by the canonical LFCA sensing machinery in EHEC to include arachidonic acid, an important bioactive lipid that is ubiquitous within host environments.

EPEC is among the leading causes of diarrheal disease worldwide. The colonization of the gut mucosa by EPEC results in actin pedestal formation at the site of bacterial attachment. These pedestals are referred to as attaching and effacing (AE) lesions. Here, we exploit the different molecular mechanisms used by EPEC to induce AE lesions on epithelial cells, showing that the effector EspFu is associated with increased bacterial attachment and enhanced epithelial colonization compared to the Tir-Nck pathway. Moreover, we also showed that actin pedestal formation can counterbalance the anti-inflammatory activity induced by EPEC, especially when driven by EspFu. Collectively, our findings provide new insights into virulence mechanisms employed by EPEC to colonize epithelial cells, as well as the host response to this enteric pathogen., The translocation of effectors into the host cell through type 3 secretion systems (T3SS) is a sophisticated strategy employed by pathogenic bacteria to subvert host responses and facilitate colonization. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) utilize the Tir and EspFu (also known as TccP) effectors to remodel the host cytoskeleton, culminating in the formation of attaching and effacing (AE) lesions on enterocytes. While some EPEC strains require tyrosine phosphorylation of Tir and recruitment of the host Nck to trigger actin polymerization, EHEC and certain EPEC strains, whose Tir is not phosphorylated, rely on the effector EspFu for efficient actin remodeling. Here, we investigated the role played by Tir-Nck and Tir-EspFu actin polymerization pathways during the infection of epithelial cells, as well as the host transcriptional response to the AE lesion formation induced by EPEC. We found that EspFu-mediated actin assembly promotes bacterial attachment and epithelial colonization more efficiently than Tir-Nck. Moreover, we showed that both actin polymerization mechanisms can activate inflammatory pathways and reverse the anti-inflammatory response induced by EPEC in epithelial cells. However, this activity is remarkably more evident in infections with EspFu-expressing EPEC strains. This study demonstrates the complex interactions between effector-mediated actin remodeling and inflammation. Different strains carry different combinations of these two effectors, highlighting the plasticity of pathogenic E. coli enteric infections.

Pathogens sense and respond to several small molecules within the GI tract to modulate expression of their virulence repertoire. Indole is a signaling molecule produced by the gut microbiota. Here we show that indole concentrations are higher in the lumen, where the microbiota is present, than in the intestinal tissue. The enteric pathogens EHEC and C. rodentium sense indole to downregulate expression of their virulence genes, as a read-out of the luminal compartment. We also identified the bacterial membrane-bound HK CpxA as an indole sensor. This regulation ensures that EHEC and C. rodentium express their virulence genes only at the epithelial lining, which is the niche they colonize., Microbial establishment within the gastrointestinal (GI) tract requires surveillance of the gut biogeography. The gut microbiota coordinates behaviors by sensing host- or microbiota-derived signals. Here we show for the first time that microbiota-derived indole is highly prevalent in the lumen compared to the intestinal tissue. This difference in indole concentration plays a key role in modulating virulence gene expression of the enteric pathogens enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium. Indole decreases expression of genes within the locus of enterocyte effacement (LEE) pathogenicity island, which is essential for these pathogens to form attaching and effacing (AE) lesions on enterocytes. We synthetically altered the concentration of indole in the GI tracts of mice by employing mice treated with antibiotics to deplete the microbiota and reconstituted with indole-producing commensal Bacteroides thetaiotaomicron (B. theta) or a B. theta ΔtnaA mutant (does not produce indole) or by engineering an indole-producing C. rodentium strain. This allowed us to assess the role of self-produced versus microbiota-produced indole, and the results show that decreased indole concentrations promote bacterial pathogenesis, while increased levels of indole decrease bacterial virulence gene expression. Moreover, we identified the bacterial membrane-bound histidine sensor kinase (HK) CpxA as an indole sensor. Enteric pathogens sense a gradient of indole concentrations in the gut to probe different niches and successfully establish an infection.

This work reveals a complex and multifaceted interaction between a human gut commensal, Enterococcus faecalis, and a pathogen, enterohemorrhagic E. coli. We demonstrate that E. faecalis enhances expression of the enterohemorrhagic E. coli type III secretion system and that this effect likely depends on cell contact between the commensal and the pathogen. Additionally, the GelE protease secreted by E. faecalis cleaves a critical structural component of the EHEC type III secretion system. In agreement with previous studies, we find that this cleavage actually increases effector protein delivery into host cells by the secretion system. This work demonstrates that commensal bacteria can significantly shape expression and activity of pathogen virulence factors, which may ultimately shape the progression of disease., The gut microbiota can significantly impact invading pathogens and the disease they cause; however, many of the mechanisms that dictate commensal-pathogen interactions remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a potentially lethal human intestinal pathogen that uses microbiota-derived molecules as cues to efficiently regulate virulence factor expression. Here, we investigate the interaction between EHEC and Enterococcus faecalis, a common human gut commensal, and show that E. faecalis affects both expression and activity of the EHEC type III secretion system (T3SS) via two distinct mechanisms. First, in the presence of E. faecalis there is increased transcription of genes encoding the EHEC T3SS. This leads to increased effector translocation and ultimately greater numbers of pedestals formed on host cells. The same effect was observed with several strains of enterococci, suggesting that it is a general characteristic of this group. In a mechanism separate from E. faecalis-induced transcription of the T3SS, we report that an E. faecalis-secreted protease, GelE, cleaves a critical structural component of the EHEC T3SS, EspB. Our data suggest that this cleavage actually increases effector translocation by the T3SS, supporting a model where EspB proteolysis promotes maximum T3SS activity. Finally, we report that treatment of EHEC with E. faecalis-conditioned cell-free medium is insufficient to induce increased T3SS expression, suggesting that this effect relies on cell contact between E. faecalis and EHEC. This work demonstrates a complex interaction between a human commensal and pathogen that impacts both expression and function of a critical virulence factor.

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of foodborne gastrointestinal illness. EHEC uses a specialized type III secretion system (T3SS) to form attaching and effacing lesions in the colonic epithelium and outcompete commensal gut microbiota to cause disease., Enterohemorrhagic Escherichia coli (EHEC) is a major cause of foodborne gastrointestinal illness. EHEC uses a specialized type III secretion system (T3SS) to form attaching and effacing lesions in the colonic epithelium and outcompete commensal gut microbiota to cause disease. A recent report in mBio (E. A. Cameron, M. M. Curtis, A. Kumar, G. M. Dunny, et al., mBio 9:e02204-18, 2018, https://doi.org/10.1128/mBio.02204-18) describes a new role for gut commensals in potentiating disease caused by EHEC. Proteases produced by EHEC and the prevalent human commensal Bacteroides thetaiotaomicron cleave proteins in the EHEC T3SS translocon that modulate T3SS function. B. thetaiotaomicron protease activity promotes translocation of bacterial effectors required for lesion formation. These results describe a new role for the microbiota in gastrointestinal disease that could uncover future treatments to prevent the spread of gastroenteritis.

Over half of adults experience gingivitis, a mild yet treatable form of periodontal disease caused by the overgrowth of oral microbes. Left untreated, gingivitis can progress to a more severe and irreversible disease, most commonly chronic periodontitis. While periodontal diseases are associated with a shift in the oral microbiota composition, it remains unclear how this shift impacts microbiota function early in disease progression. Here, we analyzed the transition from health to gingivitis through both 16S v4-v5 rRNA amplicon and metatranscriptome sequencing of subgingival plaque samples from individuals undergoing an experimental gingivitis treatment. Beta-diversity analysis of 16S rRNA reveals that samples cluster based on disease severity and patient but not by oral hygiene status. Significant shifts in the abundance of several genera occurred during disease transition, suggesting a dysbiosis due to development of gingivitis. Comparing taxonomic abundance with transcriptomic activity revealed concordance of bacterial diversity composition between the two quantification assays in samples originating from both healthy and diseased teeth. Metatranscriptome sequencing analysis indicates that during the early stages of transition to gingivitis, a number of virulence-related transcripts were significantly differentially expressed in individual and across pooled patient samples. Upregulated genes include those involved in proteolytic and nucleolytic processes, while expression levels of those involved in surface structure assembly and other general virulence functions leading to colonization or adaptation within the host are more dynamic. These findings help characterize the transition from health to periodontal disease and identify genes associated with early disease., IMPORTANCE Although more than 50% of adults have some form of periodontal disease, there remains a significant gap in our understanding of its underlying cause. We initiated this study in order to better characterize the progression from oral health to disease. We first analyzed changes in the abundances of specific microorganisms in dental plaque collected from teeth during health and gingivitis, the mildest form of periodontal disease. We found that the clinical score of disease and patient from whom the sample originated but not tooth brushing are significantly correlated with microbial community composition. While a number of virulence-related gene transcripts are differentially expressed in gingivitis samples relative to health, not all are increased, suggesting that the overall activity of the microbiota is dynamic during disease transition. Better understanding of which microbes are present and their function during early periodontal disease can potentially lead to more targeted prophylactic approaches to prevent disease progression.

Enteroaggregative Escherichia coli (EAEC) is an important diarrheal pathogen and a cause of both acute and chronic diarrhea. It is a common cause of pediatric bacterial diarrhea in developing countries. Despite its discovery in 1987, the intestinal tropism of the pathogen remains unknown. Cell lines used to study EAEC adherence include the HEp-2, T-84, and Caco-2 lines, but they exhibit abnormal metabolism and large variations in gene expression. Animal models either do not faithfully manifest human clinical symptoms or are cumbersome and expensive. Using human intestinal enteroids derived from all four segments of the human intestine, we find that EAEC demonstrates aggregative adherence to duodenal and ileal enteroids, with donor-driven differences driving a sheet-like and layered pattern. This contrasts with the colon, where segment-specific tropisms yielded a mesh-like adherence pattern dominated by interconnecting filaments. Very little to no aggregative adherence to jejunal enteroids was observed, regardless of the strain or donor, in contrast to a strong duodenal association across all donors and strains. These unique patterns of intestinal segment- or donor-specific adherence, but not the overall numbers of associated bacteria, were dependent on the major subunit protein of aggregative adherence fimbriae II (AafA), implying that the morphology of adherent clusters and the overall intestinal cell association of EAEC occur by different mechanisms. Our results suggest that we must give serious consideration to inter- and intrapatient variations in what is arguably the first step in pathogenesis, that of adherence, when considering the clinical manifestation of these infections. IMPORTANCE EAEC is a leading cause of pediatric bacterial diarrhea and a common cause of diarrhea among travelers and immunocompromised individuals. Heterogeneity in EAEC strains and lack of a good model system are major roadblocks to the understanding of its pathogenesis. Utilizing human intestinal enteroids to study the adherence of EAEC, we demonstrate that unique patterns of adherence are largely driven by unidentified factors present in different intestinal segments and from different donors. These patterns are also dependent on aggregative adherence fimbriae II encoded by EAEC. These results imply that we must also consider the contribution of the host to understand the pathogenesis of EAEC-induced inflammation and diarrhea.

Ethanolamine (EA) is a valuable source of carbon and/or nitrogen for bacteria capable of its catabolism. Because it is derived from the membrane phospholipid phosphatidylethanolamine, it is particularly prevalent in the gastrointestinal tract, which is membrane rich due to turnover of the intestinal epithelium and the resident microbiota. Intriguingly, many gut pathogens carry the eut (ethanolamine utilization) genes. EA utilization has been studied for about 50 years, with most of the early work occurring in just a couple of species of Enterobacteriaceae . Once the metabolic pathways and enzymes were characterized by biochemical approaches, genetic screens were used to map the various activities to the eut genes. With the rise of genomics, the diversity of bacteria containing the eut genes and surprising differences in eut gene content were recognized. Some species contain nearly 20 genes and encode many accessory proteins, while others contain only the core catabolic enzyme. Moreover, the eut genes are regulated by very different mechanisms, depending on the organism and the eut regulator encoded. In the last several years, exciting progress has been made in elucidating the complex regulatory mechanisms that govern eut gene expression. Furthermore, a new appreciation for how EA contributes to infection and colonization in the host is emerging. In addition to providing an overview of EA-related biology, this minireview will give special attention to these recent advances.

The growing ubiquity of electronic devices is increasingly consuming substantial energy and rare resources for materials fabrication, as well as creating expansive volumes of toxic waste. This is not sustainable. Electronic biological materials (e-biologics) that are produced with microbes, or designed with microbial components as the guide for synthesis, are a potential green solution. Some e-biologics can be fabricated from renewable feedstocks with relatively low energy inputs, often while avoiding the harsh chemicals used for synthesizing more traditional electronic materials. Several are completely free of toxic components, can be readily recycled, and offer unique features not found in traditional electronic materials in terms of size, performance, and opportunities for diverse functionalization. An appropriate investment in the concerted multidisciplinary collaborative research required to identify and characterize e-biologics and to engineer materials and devices based on e-biologics could be rewarded with a new “green age” of sustainable electronic materials and devices.

The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia , require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an “injectisome” conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia , and EspC, an autotransporter protein secreted by enteropathogenic E. coli , require a functional T3SS for host cell translocation. Both proteins have an intermediate extracellular step before their T3SS-dependent translocation. Here, we show an alternative delivery mechanism for these extracellularly secreted virulence factors that are then incorporated into the T3SS to enter the cells; this novel mechanism coexists with but diverges from the canonical injection model that involves the passage of the protein inside the injectisome.

The biogeography of the gut is diverse in its longitudinal axis, as well as within specific microenvironments. Differential oxygenation and nutrient composition drive the membership of microbial communities in these habitats. Moreover, enteric pathogens can orchestrate further modifications to gain a competitive advantage toward host colonization. These pathogens are versatile and adept when exploiting the human colon. They expertly navigate complex environmental cues and interkingdom signaling to colonize and infect their hosts. Here we demonstrate how enterohemorrhagic Escherichia coli (EHEC) uses three sugar-sensing transcription factors, Cra, KdpE, and FusR, to exquisitely regulate the expression of virulence factors associated with its type III secretion system (T3SS) when exposed to various oxygen concentrations. We also explored the effect of mucin-derived nonpreferred carbon sources on EHEC growth and expression of virulence genes. Taken together, the results show that EHEC represses the expression of its T3SS when oxygen is absent, mimicking the largely anaerobic lumen, and activates its T3SS when oxygen is available through Cra. In addition, when EHEC senses mucin-derived sugars heavily present in the O-linked and N-linked glycans of the large intestine, virulence gene expression is initiated. Sugars derived from pectin, a complex plant polysaccharide digested in the large intestine, also increased virulence gene expression. Not only does EHEC sense host- and microbiota-derived interkingdom signals, it also uses oxygen availability and mucin-derived sugars liberated by the microbiota to stimulate expression of the T3SS. This precision in gene regulation allows EHEC to be an efficient pathogen with an extremely low infectious dose., IMPORTANCE Enteric pathogens have to be crafty when interpreting multiple environmental cues to successfully establish themselves within complex and diverse gut microenvironments. Differences in oxygen tension and nutrient composition determine the biogeography of the gut microbiota and provide unique niches that can be exploited by enteric pathogens. EHEC is an enteric pathogen that colonizes the colon and causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome worldwide. It has a very low infectious dose, which requires it to be an extremely effective pathogen. Hence, here we show that EHEC senses multiple sugar sources and oxygen levels to optimally control the expression of its virulence repertoire. This exquisite regulatory control equips EHEC to sense different intestinal compartments to colonize the host.

Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium. Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine β-hydroxylase knockout (Dbh−/−) mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a noninvasive pathogen., IMPORTANCE The epinephrine and norepinephrine neurotransmitters play important roles in gut physiology and motility. Of note, epinephrine and norepinephrine play a central role in stress responses in mammals, and stress has profound effects on GI function. Bacterial enteric pathogens exploit these neurotransmitters as signals to coordinate the regulation of their virulence genes. The bacterial QseC and QseE adrenergic sensors are at the center of this regulatory cascade. C. rodentium is a noninvasive murine pathogen with a colonization mechanism similar to that of EHEC, enabling the investigation of host signals in mice. The presence of these neurotransmitters in the gut is necessary for C. rodentium to fully activate its virulence program, in a QseC/QseE-dependent manner, to successfully colonize its murine host. Our study data provide the first example of epinephrine and norepinephrine signaling within the gut to stimulate infection by a bacterial pathogen in a natural animal infection.

Bacteria engage in chemical signaling, termed quorum sensing (QS), to mediate intercellular communication, mimicking multicellular organisms. The LuxR family of QS transcription factors regulates gene expression, coordinating population behavior by sensing endogenous acyl homoserine lactones (AHLs). However, some bacteria (such as Escherichia coli) do not produce AHLs. These LuxR orphans sense exogenous AHLs but also regulate transcription in the absence of AHLs. Importantly, this AHL-independent regulatory mechanism is still largely unknown. Here we present several structures of one such orphan LuxR-type protein, SdiA, from enterohemorrhagic E. coli (EHEC), in the presence and absence of AHL. SdiA is actually not in an apo state without AHL but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life and serves as an energy source, signaling molecule, and substrate for membrane biogenesis. While exogenous AHL renders to SdiA higher stability and DNA binding affinity, OCL may function as a chemical chaperone placeholder that stabilizes SdiA, allowing for basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides crucial mechanistic insights into the ligand regulation of AHL-dependent and -independent function of LuxR-type proteins. Importantly, in addition to its contribution to basic science, this work has implications for public health, inasmuch as the SdiA signaling system aids the deadly human pathogen EHEC to adapt to a commensal lifestyle in the gastrointestinal (GI) tract of cattle, its main reservoir. These studies open exciting and novel avenues to control shedding of this human pathogen in the environment., IMPORTANCE Quorum sensing refers to bacterial chemical signaling. The QS acyl homoserine lactone (AHL) signals are recognized by LuxR-type receptors that regulate gene transcription. However, some bacteria have orphan LuxR-type receptors and do not produce AHLs, sensing them from other bacteria. We solved three structures of the E. coli SdiA orphan, in the presence and absence of AHL. SdiA with no AHL is not in an apo state but is regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL). OCL is ubiquitously found in prokaryotes and eukaryotes and is a phospholipid precursor for membrane biogenesis and a signaling molecule. While AHL renders to SdiA higher stability and DNA-binding affinity, OCL functions as a chemical chaperone placeholder, stabilizing SdiA and allowing for basal activity. Our studies provide crucial mechanistic insights into the ligand regulation of SdiA activity.

Remodeling of the host cytoskeleton is a common strategy employed by bacterial pathogens. Although there is vigorous investigation of the cell biology underlying these bacterially mediated cytoskeleton modifications, knowledge of the plasticity and dynamics of the bacterial signaling networks that regulate the expression of genes necessary for these phenotypes is lacking. Enterohemorrhagic Escherichia coli attaches to enterocytes, forming pedestal-like structures. Pedestal formation requires the expression of the locus-of-enterocyte-effacement (LEE) and espFu genes. The LEE encodes a molecular syringe, a type III secretion system (T3SS) used by pathogens to translocate effectors such as EspFu into the host cell. By using a combination of genetic, biochemical, and cell biology approaches, we show that pedestal formation relies on posttranscriptional regulation by two small RNAs (sRNAs), GlmY and GlmZ. The GlmY and GlmZ sRNAs are unique; they have extensive secondary structures and work in concert. Although these sRNAs may offer unique insights into RNA and posttranscriptional biology, thus far, only one target and one mechanism of action (exposure of the ribosome binding site from the glmS gene to promote its translation) has been described. Here we uncovered new targets and two different molecular mechanisms of action of these sRNAs. In the case of EspFu expression, they promote translation by cleavage of the transcript, while in regard to the LEE, they promote destabilization of the mRNA. Our findings reveal that two unique sRNAs act in concert through different molecular mechanisms to coordinate bacterial attachment to mammalian cells., IMPORTANCE Pathogens evolve by horizontal acquisition of pathogenicity islands. We describe here how two sRNAs, GlmY and GlmZ, involved in cellular metabolism and cellular architecture, through the posttranscriptional control of GlmS (the previously only known target of GlmY and GlmZ), which controls amino sugar synthesis, have been coopted to modulate the expression of virulence. These sRNAs quickly allow for plasticity in gene expression in order for enterohemorrhagic Escherichia coli to fine-tune the expression of its complex type III secretion machinery and its effectors to promote bacterial attachment and subsequent actin rearrangement on host cells. Pedestal formation is a very dynamic process. Many of the genes necessary for pedestal formation are located within the same operon to evolutionarily guarantee that they are inherited together. However, it is worth noting that within these operons, several genes need to yield more proteins than others and that these differences cannot be efficiently regulated at the transcriptional level.