Intact and mutated Shigella diguanylate cyclases increase c-di-GMP.

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.

Detection of OXA-181 Carbapenemase in Shigella flexneri.

We report the detection of OXA-181 carbapenemase in an azithromycin-resistant Shigella spp. bacteria in an immunocompromised patient. The emergence of OXA-181 in Shigella spp. bacteria raises concerns about the global dissemination of carbapenem resistance in Enterobacterales and its implications for the treatment of infections caused by Shigella bacteria.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

The Molecular Basis for Ubiquitin and Ubiquitin-like Specificities in Bacterial Effector Proteases.

Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins.

Ubiquitination and degradation of GBPs by a Shigella effector to suppress host defence.

Interferon-inducible guanylate-binding proteins (GBPs) mediate cell-autonomous antimicrobial defences. Shigella flexneri, a Gram-negative cytoplasmic free-living bacterium that causes bacillary dysentery, encodes a repertoire of highly similar type III secretion system effectors called invasion plasmid antigen Hs (IpaHs). IpaHs represent a large family of bacterial ubiquitin-ligases, but their function is poorly understood. Here we show that S. flexneri infection induces rapid proteasomal degradation of human guanylate binding protein-1 (hGBP1). We performed a transposon screen to identify a mutation in the S. flexneri gene ipaH9.8 that prevented hGBP1 degradation. IpaH9.8 targets hGBP1 for degradation via Lys48-linked ubiquitination. IpaH9.8 targets multiple GBPs in the cytoplasm independently of their nucleotide-bound states and their differential function in antibacterial defence, promoting S. flexneri replication and resulting in the death of infected mice. In the absence of IpaH9.8, or when binding of GBPs to IpaH9.8 was disrupted, GBPs such as hGBP1 and mouse GBP2 (mGBP2) translocated to intracellular S. flexneri and inhibited bacterial replication. Like wild-type mice, mutant mice deficient in GBP1-3, 5 and 7 succumbed to S. flexneri infection, but unlike wild-type mice, mice deficient in these GBPs were also susceptible to S. flexneri lacking ipaH9.8. The mode of IpaH9.8 action highlights the functional importance of GBPs in antibacterial defences. IpaH9.8 and S. flexneri provide a unique system for dissecting GBP-mediated immunity.

Myristoylome profiling reveals a concerted mechanism of ARF GTPase deacylation by the bacterial protease IpaJ.

N-myristoylation is an essential fatty acid modification that governs the localization and activity of cell signaling enzymes, architectural proteins, and immune regulatory factors. Despite its importance in health and disease, there are currently no methods for reversing protein myristoylation in vivo. Recently, the Shigella flexneri protease IpaJ was found to cleave myristoylated glycine of eukaryotic proteins, yet the discriminatory mechanisms of substrate selection required for targeted demyristoylation have not yet been evaluated. Here, we performed global myristoylome profiling of cells treated with IpaJ under distinct physiological conditions. The protease is highly promiscuous among diverse N-myristoylated proteins in vitro but is remarkably specific to Golgi-associated ARF/ARL family GTPases during Shigella infection. Reconstitution studies revealed a mechanistic framework for substrate discrimination based on IpaJ's function as a GTPase "effector" of bacterial origin. We now propose a concerted model for IpaJ function that highlights its potential for programmable demyristoylation in vivo.

Shigella flexneri Diguanylate Cyclases Regulate Virulence.

Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli, and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-GMP (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. c-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri. In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae, in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation and reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes. IMPORTANCE The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance among Shigella species is on the rise. Here, we explored how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We found that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella's ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

Structural mechanism for guanylate-binding proteins (GBPs) targeting by the Shigella E3 ligase IpaH9.8.

The guanylate-binding proteins (GBPs) belong to the dynamin superfamily of GTPases and function in cell-autonomous defense against intracellular pathogens. IpaH9.8, an E3 ligase from the pathogenic bacterium Shigella flexneri, ubiquitinates a subset of GBPs and leads to their proteasomal degradation. Here we report the structure of a C-terminally truncated GBP1 in complex with the IpaH9.8 Leucine-rich repeat (LRR) domain. IpaH9.8LRR engages the GTPase domain of GBP1, and differences in the Switch II and α3 helix regions render some GBPs such as GBP3 and GBP7 resistant to IpaH9.8. Comparisons with other IpaH structures uncover interaction hot spots in their LRR domains. The C-terminal region of GBP1 undergoes a large rotation compared to previously determined structures. We further show that the C-terminal farnesylation modification also plays a role in regulating GBP1 conformation. Our results suggest a general mechanism by which the IpaH proteins target their cellular substrates and shed light on the structural dynamics of the GBPs.

Maintenance of the virulence plasmid in Shigella flexneri is influenced by Lon and two functional partitioning systems.

Members of the genus Shigella carry a large plasmid, pINV, which is essential for virulence. In Shigella flexneri, pINV harbours three toxin-antitoxin (TA) systems, CcdAB, GmvAT and VapBC that promote vertical transmission of the plasmid. Type II TA systems, such as those on pINV, consist of a toxic protein and protein antitoxin. Selective degradation of the antitoxin by proteases leads to the unopposed action of the toxin once genes encoding a TA system have been lost, such as following failure to inherit a plasmid harbouring a TA system. Here, we investigate the role of proteases in the function of the pINV TA systems and demonstrate that Lon, but not ClpP, is required for their activity during plasmid stability. This provides the first evidence that acetyltransferase family TA systems, such as GmvAT, can be regulated by Lon. Interestingly, S. flexneri pINV also harbours two putative partitioning systems, ParAB and StbAB. We show that both systems are functional for plasmid maintenance although their activity is masked by other systems on pINV. Using a model vector based on the pINV replicon, we observe temperature-dependent differences between the two partitioning systems that contribute to our understanding of the maintenance of virulence in Shigella species.

Quercetin a major biomarker of Psidium guajava L. inhibits SepA protease activity of Shigella flexneri in treatment of infectious diarrhoea.

The leaves of the plant Psidium guajava L. (Myrtaceae) has been traditionally used in treatment of various gastrointestinal disorders including diarrhoea and have also been reported for its potent antidiarrhoeal activity on various chemical induced diarrhoea models. The objective of our present study was to evaluate the potency of the leaf extract of the plant Psidium guajava (PGE) along with its major biomarker quercetin against Shigella flexneri-induced sub chronic model of infectious diarrhoea. PGE at 100, 200 and 400 mg/kg, p.o. and quercetin at 50 mg/kg, p.o. were administered to Shigella flexneri-induced diarrhoeal rats for five days and various behavioural parameters were evaluated on 1st, 3rd and 5th day of treatment. This was followed by assessment of stool water content, density of Shigella flexneri in stools and blood parameters examination. After treatment, colon and small intestine of rats was dissected and subjected to biochemical estimations, cytokine profiling, antioxidant evaluations, ion concentration determination, Na+/K+-ATPase activity and histopathology. Molecular docking studies on crystal structure of Secreted Extracellular Protein A (SepA) from Shigella flexneri with biomarker quercetin was also performed. PGE at 200 mg/kg followed by quercetin depicted maximum antidiarrhoeal potential, which was confirmed through diarrhoea score and % protection, while PGE at 400 mg/kg showed similar effect to PGE 200 mg/kg thus, the later may have ceiling effect. PGE and quercetin also significantly reduced the density of Shigella flexneri in stools, water content of stools and restored the alterations observed in blood parameters, antioxidant status and pro-inflammatory cytokines (IL-6 and TNF-α) expression. These parameters contributed in normalization of electrolyte balance, reactivation of Na+/K+-ATPase activity and repairing of epithelial tissue damage, confirmed through histopathology. Docking simulation studies revealed the role of quercetin in inactivating the protease activity of SepA, a protein secreted by Shigella, which disrupts epithelial barrier integrity during infection and also manages its signal production. Thus, the overall results confirmed the role of quercetin as a major biomarker for the observed antidiarrhoeal potential of P. guajava against Shigella flexneri induced infectious diarrhoea.

Structural analysis of Shigella flexneri bi-functional enzyme HisIE in histidine biosynthesis.

Histidine biosynthesis, which is absent in animals, was shown to be highly conserved among gram-negative bacteria, thus making it an attractive target for antibiotic design. There are many fusion forms of enzymes in the histidine biosynthetic pathway and people still have limited knowledge about their domain organizations and catalytic mechanisms, due to the lack of structural information. Here we report the first crystal structure of Shigella flexneri bi-functional enzyme HisIE (SfHisIE) that functions in the 2nd and 3rd steps in the histidine biosynthetic pathway. This structure shows that HisIE exists as dimers with two loops (fusion loop) connecting the individual dimer of HisE and HisI in its N-terminus and C-terminus respectively. Our mutagenesis study shows mutations in this fusion loop are lethal for bacteria indicating the advantage of gene fusion in Histidine biosynthesis. Structural analysis revealed several highly conserved residues in the putative ligand binding grooves of HisE and HisI, showing an evolutionarily conserved catalytic mechanism shared among gram negative-bacteria.

Cell-cell propagation of NF-κB transcription factor and MAP kinase activation amplifies innate immunity against bacterial infection.

The enteroinvasive bacterium Shigella flexneri uses multiple secreted effector proteins to downregulate interleukin-8 (IL-8) expression in infected epithelial cells. Yet, massive IL-8 secretion is observed in Shigellosis. Here we report a host mechanism of cell-cell communication that circumvents the effector proteins and strongly amplifies IL-8 expression during bacterial infection. By monitoring proinflammatory signals at the single-cell level, we found that the activation of the transcription factor NF-κB and the MAP kinases JNK, ERK, and p38 rapidly propagated from infected to uninfected adjacent cells, leading to IL-8 production by uninfected bystander cells. Bystander IL-8 production was also observed during Listeria monocytogenes and Salmonella typhimurium infection. This response could be triggered by recognition of peptidoglycan and is mediated by gap junctions. Thus, we have identified a mechanism of cell-cell communication that amplifies innate immunity against bacterial infection by rapidly spreading proinflammatory signals via gap junctions to yet uninfected cells.

Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ.

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.

Characterization of the complete sequences and stability of plasmids carrying the genes aac(6')-Ib-cr or qnrS in Shigella flexneri in the Hangzhou area of China.

The aim of this study was to explore the fluoroquinolone resistance mechanism of aac (6')-Ib-cr and qnrS gene by comparing complete sequences and stability of the aac(6')-Ib-cr- and qnrS-positive plasmids from Shigella isolates in the Hangzhou area of China. The complete sequences of four newly acquired plasmids carrying aac(6')-Ib-cr or qnrS were compared with those of two plasmids obtained previously and two similar reference Escherichia coli plasmids. The results showed that the length, antibiotic resistance genes and genetic environment were different among the plasmids. Moreover, the plasmid stability of three wild-type isolates and five plasmid transformants carrying aac(6')-Ib-cr and/or qnrS was measured in vitro, and all eight isolates were found to have lost their aac(6')-Ib-cr- or qnrS-positive plasmids to a different extent at different stages. When the plasmids were electroporated into Shigella flexneri or they lost positive plasmids, the MICs of ciprofloxacin increased or decreased two- to eightfold for aac(6')-Ib-cr-positive plasmids and 16- to 32-fold for qnrS-positive plasmids. To our knowledge, this is the first report comparing the complete sequences and describing stability for the aac(6')-Ib-cr- and qnrS-positive plasmids from Shigella isolates.

Midori-ishi Cyan/monomeric Kusabira-Orange-based fluorescence resonance energy transfer assay for characterization of various E3 ligases.

Many bacterial pathogens hijack the host ubiquitin system for their own benefit by delivering effectors with ubiquitin ligase (E3) into host cells via the type III secretion system. Therefore, screening for small compounds that selectively inhibit bacterial but not mammalian E3 ligases is a promising strategy for identifying molecules that could substitute for antibiotics. To facilitate high-throughput screening for bacterial E3 ligase inhibitors, we developed a MiCy/mKO (Midori-ishi Cyan/monomeric Kusabira-Orange)-based FRET (fluorescence resonance energy transfer) assay and validated it on Shigella IpaH E3 ligase effectors. We showed the feasibility of using the MiCy/mKO-based FRET assay to identify the most appropriate ubiquitin-conjugating enzymes (E2s) and determine the lysine specificity of a given E3, both hallmarks of E3 activity. Furthermore, we showed the usefulness of the FRET assay in characterizing mammalian E3 ligases, such as TNF receptor-associated factor 6 (TRAF6) and mouse double minute 2 homologue (MDM2). In addition, we confirmed the feasibility of determining the efficiency of inhibition of E3 ligase activity using inhibitors of E1 ubiquitin-activating enzymes, such as UBE1-41, by measuring the IC50 . Based on these results, we concluded that the MiCy/mKO-based FRET assay is useful for characterizing E3 enzyme activity, as well as for high-throughput E3 inhibitor screening.

Horizontal gene transfer of a Chlamydial tRNA-guanine transglycosylase gene to eukaryotic microbes.

tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes.

Alteration in apyrase enzyme attenuated virulence of Shigella flexneri.

Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing mitochondrial damage and host cell death. In this study we constructed an apy mutant of Shigella flexneri by insertional activation using a kanamycin resistant gene cassette. The wild type apy gene of S. flexneri 2a was PCR amplified, cloned and mutated with insertion of kanamycin resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type S. flexneri 2a by conjugation with Escherichia coli. After undergoing homologous recombination, the wild apy gene was deleted from the construct using the sucrose selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the guinea pig keratoconjunctivitis model. The strain was Sereny negative and it elicited a significant protection following challenge with wild S. flexneri strain. This apy mutant strain will form a base for the development of a vaccine target for shigellosis.

Novel mutations in quinolone resistance-determining regions of gyrA, gyrB, parC and parE in Shigella flexneri clinical isolates from eastern Chinese populations between 2001 and 2011.

The aim of this study was to evaluate the prevalence of fluoroquinolone resistance and mechanisms of selected fluoroquinolone resistance in Shigella flexneri isolates. A total of 624 S. flexneri strains isolated between 2001 and 2011 in Jiangsu Province of China were analysed for their fluoroquinolone susceptibility. The quinolone resistance-determining region of gyrA, gyrB, parC and parE were amplified and sequenced. In general, 90.5 % of S. flexneri exhibited resistance to nalidixic acid. The mean norfloxacin resistance rate was 22.4 % during the 11 years from 2001 to 2011 (6.4 % from 2001 to 2005 and 36.8 % from 2006 to 2011). Sequencing of gyrA, gyrB, parC and parE genes of all S. flexneri isolates showed that the mutation rate was as high as 93.9 %. In addition, 91.8 % and 92.3 % of S. flexneri harboured mutations in gyrA and parC, respectively. About 35.2 % of S. flexneri isolates susceptible to nalidixic acid contained mutations. Meanwhile, mutations were detected in 91.2 % of norfloxacin-susceptible strains, and almost all S. flexneri isolates resistant to fluoroquinolone contained mutations. To the best of our knowledge, this is the first study reporting the occurrence of point mutations Asn57Lys and His80Pro in gyrA and Ala85Thr, Asp111His and Ser129Pro in parC. Emerging fluoroquinolone resistance with a significantly high mutation rate of the gyrA and parC genes in S. flexneri in Jiangsu Province deserves attention, and monitoring antibiotic susceptibility is important for the effective management of S. flexneri infections.

Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants.

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability.