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CTX-Ms are encoded by bla_CTX-M genes and are widely distributed extended-spectrum β-lactamases (ESBLs). They are the most important antimicrobial resistance (AMR) mechanism to β-lactam antibiotics in the Enterobacteriaceae. However, the role of transmissible AMR plasmids in the dissemination of bla_CTX-M genes has scarcely been studied in Africa where the burden of AMR is high and rapidly spreading. In this study, AMR plasmid transmissibility, replicon types and addiction systems were analysed in CTX-M-producing Escherichia coli clinical isolates in Ethiopia with a goal to provide molecular insight into mechanisms underlying such high prevalence and rapid dissemination. Of 100 CTX-Ms-producing isolates obtained from urine (84), pus (10) and blood (6) from four geographically distinct healthcare settings, 75% carried transmissible plasmids encoding for CTX-Ms, with CTX-M-15 being predominant (n = 51). Single IncF plasmids with the combination of F-FIA-FIB (n = 17) carried the bulk of bla_CTX-M-15 genes. In addition, IncF plasmids were associated with multiple addiction systems, ISEcp1 and various resistance phenotypes for non-cephalosporin antibiotics. Moreover, IncF plasmid carriage is associated with the international pandemic E. coli ST131 lineage. Furthermore, several CTX-M encoding plasmids were associated with serum survival of the strains, but less so with biofilm formation. Hence, both horizontal gene transfer and clonal expansion may contribute to the rapid and widespread distribution of bla_CTX-M genes among E. coli populations in Ethiopian clinical settings. This information is relevant for local epidemiology and surveillance, but also for global understanding of the successful dissemination of AMR gene carrying plasmids.

Translocon pores formed in the eukaryotic cell membrane by a type III secretion system facilitate the translocation of immune-modulatory effector proteins into the host cell interior. The YopB and YopD proteins produced and secreted by pathogenic Yersinia spp. harboring a virulence plasmid-encoded type III secretion system perform this pore-forming translocator function. We had previously characterized in vitro T3SS function and in vivo pathogenicity of a number of strains encoding sited-directed point mutations in yopD. This resulted in the classification of mutants into three different classes based upon the severity of the phenotypic defects. To investigate the molecular and functional basis for these defects, we explored the effectiveness of RAW 264.7 cell line to respond to infection by representative YopD mutants of all three classes. Signature cytokine profiles could separate the different YopD mutants into distinct categories. The activation and suppression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to alter anti-phagocytosis and programmed cell death pathways. These analyses demonstrated that sub-optimal translocon pores impact the extent and magnitude of host cell responsiveness, and this limits the capacity of pathogenic Yersinia spp. to fortify against attack by both early and late arms of the host innate immune response.

YscX was discovered as an essential part of the Yersinia type III secretion system about 20 years ago. It is required for substrate secretion and is exported itself. Despite this central role, its precise function and mode of action remain unknown. In order to address this knowledge gap, this present study refocused attention on YscX to build on the recent advances in the understanding of YscX function. Our experiments identified an N-terminal secretion domain in YscX promoting its secretion, with the first five codons constituting a minimal signal capable of promoting secretion of the signal less β-lactamase reporter. Replacing the extreme YscX N-terminus with known secretion signals of other Ysc-Yop substrates revealed that the YscX N-terminal segment contains non-redundant information needed for YscX function. Further, both in cis deletion of the YscX N-terminus in the virulence plasmid and ectopic expression of epitope-tagged YscX variants again lead to stable YscX production but not type III secretion of Yop effector proteins. Mislocalisation of the needle components, SctI and SctF, accompanied this general defect in Yops secretion. Hence, a coupling exists between YscX secretion permissiveness and the assembly of an operational secretion system.

The treatment of invasive Escherichia coli infections is a challenge because of the emergence and rapid spread of multidrug resistant strains. Particular problems are those strains that produce extended spectrum β-lactamases (ESBL's). Although the global characterization of these enzymes is advanced, knowledge of their molecular basis among clinical E. coli isolates in Ethiopia is extremely limited. This study intends to address this knowledge gap. The study combines antimicrobial resistance profiling and molecular epidemiology of ESBL genes among 204 E. coli clinical isolates collected from patient urine, blood, and pus at four geographically distinct health facilities in Ethiopia. All isolates exhibited multidrug resistance, with extensive resistance to ampicillin and first to fourth line generation cephalosporins and sulfamethoxazole-trimethoprim and ciprofloxacin. Extended spectrum β-lactamase genes were detected in 189 strains, and all but one were positive for CTX-Ms β-lactamases. Genes encoding for the group-1 CTX-Ms enzymes were most prolific, and CTX-M-15 was the most common ESBL identified. Group-9 CTX-Ms including CTX-M-14 and CTX-27 were detected only in 12 isolates and SHV ESBL types were identified in just 8 isolates. Bacterial typing revealed a high amount of strains associated with the B2 phylogenetic group. Crucially, the international high risk clones ST131 and ST410 were among the sequence types identified. This first time study revealed a high prevalence of CTX-M type ESBL's circulating among E. coli clinical isolates in Ethiopia. Critically, they are associated with multidrug resistance phenotypes and high-risk clones first characterized in other parts of the world. 

Various Gram-negative bacteria utilize type III secretion system (T3SS) to deliver effectors into eukaryotic host cells and establish mutualistic or pathogenic interactions. An example is the Ysc-Yop T3SS of pathogenic Yersinia species. The T3SS resembles a molecular syringe with a wide cylindrical membrane-spanning basal body that scaffolds a hollow extracellular needle with a pore-forming translocon complex crowned at the needle tip. Together they form a continuous conduit between bacteria and host cells that allow delivery of effector proteins. 

Dedicated actions of cytoplasmic chaperones, regulators and components of the cytoplasmic complex orchestrates hierarchical assembly of T3SS. On the basis of secretion hierarchy, proteins can be categorized as ‘early’ needle complex proteins, ‘middle’ translocators and ‘late’ Yop effectors. However, how the system recognizes, prepares and mediates temporal delivery of T3S substrates is not fully understood. Herein, we have investigated the roles of YscX and YscY (present specifically in the Ysc family of T3SS), as well as YopN-TyeA (broadly distributed among T3SS families) to provide a better understanding of some of the molecular mechanisms governing spatiotemporal control of T3SS assembly.

Despite reciprocal YscX-YscY binary and YscX-YscY-SctV ternary interactions between the member proteins, functional interchangeability in Yersinia was not successful. This revealed YscX and YscY must perform functions unique to Yersinia T3SS. Defined domain swapping and site-directed mutagenesis identified two highly conserved cysteine residues important for YscX function. Moreover, the N-terminal region of YscX harboured an independent T3S signal. Manipulating the YscX N-terminus by exchanging it with equivalent secretion signals from different T3S substrates abrogated T3S activity. This was explained by the need for the YscX N-terminus to correctly localize and/or assemble the ‘early’ SctI inner adapter and SctF needle protein. Therefore, N-terminal YscX performs dual functions; one as a secretion signal and the other as a structural signal to control early stage assembly of T3SS.

In Ysc-Yop T3SS, YopN-TyeA complex is involved in the later stage of T3SS assembly, inhibiting Yops secretion until host cell contact is achieved. Analysis of the YopN C-terminus identified a specific domain stretching 279-287 critical for regulating Ysc-Yop T3SS activity. The regulation was mediated by specific hydrophobic contacts between W₂₇₉ of YopN and F₈ of TyeA.

In conclusion, this work has provided novel molecular mechanisms regarding  the spatiotemporal assembly of T3SS. While the N-terminal region of YscX contributes to the early stage of T3SS assembly, the C-terminal region of YopN is critical for regulating Ysc-Yop activity at a later stage of T3SS assembly.

The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.

Type III secretion systems harbored by several Gram-negative bacteria are often used to deliver host-modulating effectors into infected eukaryotic cells. About 20 core proteins are needed for assembly of a secretion apparatus. Several of these proteins are genetically and functionally conserved in type III secretion systems of bacteria associated with invertebrate or vertebrate hosts. In the Ysc family of type III secretion systems are two poorly characterized protein families, the YscX family and the YscY family. In the plasmid-encoded Ysc-Yop type III secretion system of human pathogenic Yersinia species, YscX is a secreted substrate while YscY is its non-secreted cognate chaperone. Critically, neither an yscX nor yscY null mutant of Yersinia is capable of type III secretion. In this study, we show that the genetic equivalents of these proteins produced as components of other type III secretion systems of Pseudomonas aeruginosa (PscX and PscY), Aeromonas species (AscX and AscY), Vibrio species (VscX and VscY), and Photorhabdus luminescens (SctX and SctY) all possess an ability to interact with its native cognate partner and also establish cross-reciprocal binding to non-cognate partners as judged by a yeast two-hybrid assay. Moreover, a yeast three-hybrid assay also revealed that these heterodimeric complexes could maintain an interaction with YscV family members, a core membrane component of all type III secretion systems. Despite maintaining these molecular interactions, only expression of the native yscX in the near full-length yscX deletion and native yscY in the near full-length yscY deletion were able to complement for their general substrate secretion defects. Hence, YscX and YscY must have co-evolved to confer an important function specifically critical for Yersinia type III secretion.

Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W₂₇₉ functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN_W279G mutant lost all ability to bind TyeA. The TyeA residue F₈ was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor sigma(E). In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the sigma(E)-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.

Pathogenic Yersinia spp. utilizes the Ysc-Yop type III secretion system to targetYop effector proteins into the cytosol of host immune cells. Internalizedeffectors alter specific signaling pathways to neutralize immune cell-dependentphagocytosis, killing and pro-inflammatory responsiveness. This enablesextracellular bacterial multiplication and survival in immune tissue. Central tothe temporal control of Yop type III secretion is the regulator YopN. Incomplex with TyeA, YopN acts to plug the inner face of the type III secretionchannel, denying entry to other Yop substrates until after YopN has beensecreted. A +1 frameshift event in the 3-prime end of yopN results in thesynthesis of a singular secreted YopN-TyeA polypeptide chimera that retainssome regulatory function. As the C-terminal coding sequence of YopN in thishybrid product differs greatly from native sequence, we used site-directedmutagenesis to determine the functional significance of this segment. YopNtruncated at residue 287 or containing a shuffled sequence covering 288 to 293retains full function both in vitro and in vivo. Thus, the extreme C-terminus isapparently superfluous to YopN function. In contrast, a YopN varianttruncated after residue 278 was completely unstable, and these bacteria hadlost all control of T3S activity, and failed to defend against immune cell killing.Interestingly, inclusion of a shuffled sequence from residues 279 to 287recovered some T3S control over function. Hence, the YopN segmentencompassing 279 to 287 is essential for full function, although the exact aminoacid sequence is less important.