The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae.

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir.

ChiS is a noncanonical DNA-binding hybrid sensor kinase that directly regulates the chitin utilization program in Vibrio cholerae.

Two-component signal transduction systems (TCSs) represent a major mechanism that bacteria use to sense and respond to their environment. Prototypical TCSs are composed of a membrane-embedded histidine kinase, which senses an environmental stimulus and subsequently phosphorylates a cognate partner protein called a response regulator that regulates gene expression in a phosphorylation-dependent manner. Vibrio cholerae uses the hybrid histidine kinase ChiS to activate the expression of the chitin utilization program, which is critical for the survival of this facultative pathogen in its aquatic reservoir. A cognate response regulator for ChiS has not been identified and the mechanism of ChiS-dependent signal transduction remains unclear. Here, we show that ChiS is a noncanonical membrane-embedded one-component system that can both sense chitin and directly regulate gene expression via a cryptic DNA binding domain. Unlike prototypical TCSs, we find that ChiS DNA binding is diminished, rather than stimulated, by phosphorylation. Finally, we provide evidence that ChiS likely activates gene expression by directly recruiting RNA polymerase. This work addresses the mechanism of action for a major transcription factor in V. cholerae and highlights the versatility of signal transduction systems in bacterial species.

Campylobacter coli infection causes spinal epidural abscess with Guillain-Barré syndrome: a case report.

BACKGROUND: Guillain-Barré syndrome (GBS) and spinal epidural abscess (SEA) are known as mimics of each other because they present with flaccid paralysis following an infection; however, they differ in the main causative bacteria. Nevertheless, the two diseases can occur simultaneously if there is a preceding Campylobacter infection. Here, we report the first case of SEA with GBS following Campylobacter coli infection. CASE PRESENTATION: A 71-year-old Japanese man presented with progressive back pain and paralysis of the lower limbs following enteritis. Magnetic resonance imaging showed a lumbar epidural abscess that required surgical decompression; therefore, surgical drainage was performed. Blood cultures revealed the presence of C. coli. Despite surgery, the paralysis progressed to the extremities. Nerve conduction studies led to the diagnosis of GBS. Anti-ganglioside antibodies in the patient suggested that GBS was preceded by Campylobacter infection. Intravascular immunoglobulin therapy attenuated the progression of the paralysis. CONCLUSIONS: We report a case of SEA and GBS following Campylobacter infection. A combination of the two diseases is rare; however, it could occur if the preceding infection is caused by Campylobacter spp. If a cause is known but the patient does not respond to the corresponding treatment, it is important to reconsider the diagnosis based on the medical history.

Molecular mechanisms of Streptococcus pneumoniae-targeted autophagy via pneumolysin, Golgi-resident Rab41, and Nedd4-1-mediated K63-linked ubiquitination.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae.

In Vibrio cholerae, the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326-347, 2014, https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription of tfoR, encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned off tfoR::lacZ expression but possessed intact chiS and tfoS genes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIAglc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIAglc inactivated natural competence and tfoR transcription. Chitin-induced expression of the chb operon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIAglc Furthermore, the regulation of tfoR and chb expression by EIIAglc was dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIAglc These results define a previously unknown connection between the PTS and chitin signaling pathways in V. cholerae and suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.IMPORTANCE The EIIAglc protein of the PTS coordinates a wide variety of physiological functions with carbon availability. In this report, we describe an unexpected association of chitin-activated signaling pathways in V. cholerae with EIIAglc The signaling pathways are governed by the chitin-responsive TCS sensor kinase ChiS and lead to the induction of chitin utilization and natural competence. We show that dephosphorylated EIIAglc inhibits both signaling pathways in a ChiS-dependent manner. This inhibition is different from classical catabolite repression that is caused by lowered levels of cyclic AMP. This work represents a newly identified connection between the PTS and chitin signaling pathways in V. cholerae and suggests a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.

Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae.

In Vibrio cholerae, 41 chitin-inducible genes, including the genes involved in natural competence for DNA uptake, are governed by the orphan two-component system (TCS) sensor kinase ChiS. However, the mechanism by which ChiS controls the expression of these genes is currently unknown. Here, we report the involvement of a novel transcription factor termed 'TfoS' in this process. TfoS is a transmembrane protein that contains a large periplasmic domain and a cytoplasmic AraC-type DNA-binding domain, but lacks TCS signature domains. Inactivation of tfoS abolished natural competence as well as transcription of the tfoR gene encoding a chitin-induced small RNA essential for competence gene expression. A TfoS fragment containing the DNA-binding domain specifically bound to and activated transcription from the tfoR promoter. Intracellular TfoS levels were unaffected by disruption of chiS and coexpression of TfoS and ChiS in Escherichia coli recovered transcription of the chromosomally integrated tfoR::lacZ gene, suggesting that TfoS is post-translationally modulated by ChiS during transcriptional activation; however, this regulation persisted when the canonical phosphorelay residues of ChiS were mutated. The results presented here suggest that ChiS operates a chitin-induced non-canonical signal transduction cascade through TfoS, leading to transcriptional activation of tfoR.

RodZ regulates the post-transcriptional processing of the Shigella sonnei type III secretion system.

The expression of the type III secretion system-a main determinant of virulence in Shigella-is controlled by regulator cascades VirF-InvE (VirB) and CpxAR two-component system. A screen for mutants that restore virulence in the cpxA background led to the isolation of a mutant of rodZ, a cytoskeletal protein that maintains the rod-shaped morphology of bacilli. InvE is normally repressed at 30 °C because of decreased messenger RNA (mRNA) stability, but rodZ mutants markedly increase invE-mRNA stability. Importantly, the inhibition of InvE production by RodZ can be genetically separated from its role in cell-shape maintenance, indicating that these functions are distinguishable. Thus, we propose that RodZ is a new membrane-bound RNA-binding protein that provides a scaffold for post-transcriptional regulation.

Horizontal gene transfer of a genetic island encoding a type III secretion system distributed in Vibrio cholerae.

Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS-related genes were distributed among the various serogroups and pulsed-field gel electrophoresis of NotI-digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS-related genes had similar patterns. Additionally, naturally competent T3SS-negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS-related genes occurs among V. cholerae in natural ecosystems.

Identification of a chitin-induced small RNA that regulates translation of the tfoX gene, encoding a positive regulator of natural competence in Vibrio cholerae.

The tfoX (also called sxy) gene product is the central regulator of DNA uptake in the naturally competent bacteria Haemophilus influenzae and Vibrio cholerae. However, the mechanisms regulating tfoX gene expression in both organisms are poorly understood. Our previous studies revealed that in V. cholerae, chitin disaccharide (GlcNAc)2 is needed to activate the transcription and translation of V. cholerae tfoX (tfoX(VC)) to induce natural competence. In this study, we screened a multicopy library of V. cholerae DNA fragments necessary for translational regulation of tfoX(VC). A clone carrying the VC2078-VC2079 intergenic region, designated tfoR, increased the expression of a tfoX(VC)::lacZ translational fusion constructed in Escherichia coli. Using a tfoX(VC)::lacZ reporter system in V. cholerae, we confirmed that tfoR positively regulated tfoX(VC) expression at the translational level. Deletion of tfoR abolished competence for exogenous DNA even when (GlcNAc)2 was provided. The introduction of a plasmid clone carrying the tfoR(+) gene into the tfoR deletion mutant complemented the competence deficiency. We also found that the tfoR gene encodes a 102-nucleotide small RNA (sRNA), which was transcriptionally activated in the presence of (GlcNAc)2. Finally, we showed that this sRNA activated translation from tfoX(VC) mRNA in a highly purified in vitro translation system. Taking these results together, we propose that in the presence of (GlcNAc)2, TfoR sRNA is expressed to activate the translation of tfoX(VC), which leads to the induction of natural competence.

Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.

A multiplex PCR assay was developed based on atpA-sequence diversification for molecular identification of 3 major pathogenic Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. It specifically identified them from among 133 strains of various Vibrio species and other genera, and was applicable for testing seawater, suggesting its usefulness.

Stabilizing Genetically Unstable Simple Sequence Repeats in the Campylobacter jejuni Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes.

Hypermutable simple sequence repeats (SSRs) are major drivers of phase variation in Campylobacter jejuni. The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), generates extreme cell surface diversity within bacterial populations, thereby promoting adaptation to selective pressures in host environments. Therefore, genetically controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. Here, we show that natural "cotransformation" is an effective method for C. jejuni genome editing. Cotransformation is a trait of naturally competent bacteria that causes uptake/integration of multiple different DNA molecules, which has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple mutations into the genomes of these bacteria. We found that cotransformation efficiently occurred in C. jejuni. To examine the feasibility of MuGENT in C. jejuni, we "locked" different polyG SSR tracts in strain NCTC11168 (which are located in the biosynthetic CPS/LOS gene clusters) into either the ON or OFF configurations. This approach, termed "MuGENT-SSR," enabled the generation of all eight edits within 2 weeks and the identification of a phase-locked strain with a highly stable type of Penner serotyping, a CPS-based serotyping scheme. Furthermore, extensive genome editing of this strain by MuGENT-SSR identified a phase-variable gene that determines the Penner serotype of NCTC11168. Thus, MuGENT-SSR provides a platform for genetic and phenotypic engineering of genetically unstable C. jejuni, making it a reliable approach for elucidating the mechanisms underlying phase-variable expression of specific phenotypes. IMPORTANCE Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis in developed countries and occasionally progresses to the autoimmune disease Guillain-Barré syndrome. A relatively large number of hypermutable simple sequence repeat (SSR) tracts in the C. jejuni genome markedly decreases its phenotypic stability through reversible changes in the ON or OFF expression states of the genes in which they reside, a phenomenon called phase variation. Thus, controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. In this study, we developed a feasible and effective approach for genetically manipulate multiple SSR tracts in the C. jejuni genome using natural cotransformation, a trait of naturally transformable bacterial species that causes the uptake and integration of multiple different DNA molecules. This approach will greatly help to improve the genetic and phenotypic stability of C. jejuni to enable diverse applications in research and development.

LrhA positively controls the expression of the locus of enterocyte effacement genes in enterohemorrhagic Escherichia coli by differential regulation of their master regulators PchA and PchB.

Summary Genes essential for eliciting pathogenicity of enterohemorrhagic Escherichia coli are located within the locus of enterocyte effacement (LEE). Expression of LEE genes is positively regulated by paralogues PchA, PchB and PchC, which are encoded by separate loci of the chromosome. To elucidate the underlying regulatory mechanism, we screened transposon mutants exhibiting reduced expression of pchA, transcription level of which is highest among the pch genes. Here, we report that the LysR-homologue A (LrhA) positively regulated the transcription of pchA and pchB. A deletion in lrhA reduced the transcription levels of pchA and pchB to different degrees, and also reduced the expression of LEE-coded type 3-secreted protein, EspB. Expression of LrhA from a plasmid restored and markedly increased the transcription levels of pchA and pchB respectively, and highly induced EspB expression. Deletion analysis of the regulatory region showed that both promoter-proximal (-195 to +88) and promoter-distal (-418 to -392 for pchA and -391 to -375 for pchB) sequences were required for the LrhA-mediated upregulation of pchA and pchB genes. Purified His(6)-LrhA protein differentially bound to the regulatory regions of pchA/B, suggesting that direct regulation of pchA and pchB genes by LrhA in turn controls the expression of LEE genes.

The effect of radiotherapy on NKT cells in patients with advanced head and neck cancer.

BACKGROUND: Cancer immunotherapy with NKT cells is a potential new treatment strategy for advanced head and neck cancer. NKT cell therapy is promising due to its unique anti-tumor activity and higher degree of safety compared to current therapies. Radiotherapy is indispensable as a standard treatment for advanced head and neck cancer. To elucidate the possibility of using NKT cells as an adjuvant immunotherapy with radiotherapy, we examined the effect of radiotherapy on NKT cells in patients with head and neck cancer. METHODS: The number, IFN-gamma production and proliferation capacity of NKT cells were analyzed before and after 50 Gy radiation therapy in 12 patients with stage IV head and neck squamous cell carcinoma. The cytotoxic activity of NKT cells was examined in vitro. RESULTS: The number of NKT cells in the blood varied widely between patients. After radiation therapy, the population of CD3 T cells decreased significantly, while the NKT cell population remained stable. The number of NKT cells was the same after radiation therapy as before. IFN-gamma production from NKT cells collected just after radiotherapy was impaired after stimulation with exogenous ligand, but the proliferative responses of these NKT cells was enhanced in comparison to those collected before radiation therapy. Furthermore, the proliferated NKT cells displayed a significant level of anti-tumor activity. CONCLUSION: NKT cells are relatively resistant to radiation and might therefore be suitable for adjuvant immunotherapy to eradicate remnant cancer cells in patients who have undergone radiation therapy.

Regulation of lankamycin biosynthesis in Streptomyces rochei by two SARP genes, srrY and srrZ.

Transcription and complementation experiments were carried out to analyze the regulatory hierarchy of two Streptomyces antibiotic regulatory protein (SARP) genes, srrY and srrZ, in the gamma-butyrolactone (GB)-dependent regulatory cascade in Streptomyces rochei 7434AN4. The srrY gene was transcribed in the srrZ mutant, while the srrZ gene was not in the srrY mutant. The SrrY protein was specifically bound to the promoter region of srrZ, where a possible SARP binding sequence was identified 26 bp upstream of the -10 sequence. Deletion of the repeat sequences from this region abolished its SrrY binding activity. In addition, complementation of srrZ restored lankamycin production in the srrY mutant. All of these results confirm that the SARP gene srrY directly regulates expression of the second SARP gene srrZ in a positive manner. This study gave the first confirmation of direct regulation of two SARP genes in the GB-dependent regulatory cascade in Streptomyces.

Superstructure formation by RodZ hexamers of Shigella sonnei maintains the rod shape of bacilli.

The rod shape of bacilli is maintained by bacterial cytoskeletal protein MreB, an actin homolog that acts in concert with the inner membrane protein RodZ. We previously reported RodZ binds RNA to control the posttranscriptional regulation of invE (virB), which controls the type III secretion system essential for the virulence of Shigella. Here, we show that purified RodZ forms "superstructures" of high molecular mass that dissociate into a midsized "basal complex" in the presence of nonionic detergent, or to a monomer in the presence of dithiothreitol. We used mass spectrometry to show that the basal complex was a hexamer. Electrophoresis mobility shift assays combined with gel filtration detected the RNA-binding activity in fractions containing molecules larger than the basal hexamer. The superstructure was consistently detected with MreB in crude cell lysates of S. sonnei that were fractionated using gel filtration. Immunofluorescence microscopy using two different super-resolution settings showed that wild-type RodZ was distributed in cells as separate dots. Consistent with the superstructure comprising homohexamers, majority of the dots distributed among areas of discrete values. In addition, simultaneous immunodetection of MreB provided the first evidence of colocalization with RodZ as larger patch like signals. These findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.

SrrB, a Pseudo-Receptor Protein, Acts as a Negative Regulator for Lankacidin and Lankamycin Production in Streptomyces rochei.

Streptomyces rochei 7434AN4, a producer of lankacidin (LC) and lankamycin (LM), carries many regulatory genes including a biosynthesis gene for signaling molecules SRBs (srrX), an SRB receptor gene (srrA), and a SARP (Streptomyces antibiotic regulatory protein) family activator gene (srrY). Our previous study revealed that the main regulatory cascade goes from srrX through srrA to srrY, leading to LC production, whereas srrY further regulates a second SARP gene srrZ to synthesize LM. In this study we extensively investigated the function of srrB, a pseudo-receptor gene, by analyzing antibiotic production and transcription. Metabolite analysis showed that the srrB mutation increased both LC and LM production over four-folds. Transcription, gel shift, and DNase I footprinting experiments revealed that srrB and srrY are expressed under the SRB/SrrA regulatory system, and at the later stage, SrrB represses srrY expression by binding to the promoter region of srrY. These findings confirmed that SrrB acts as a negative regulator of the activator gene srrY to control LC and LM production at the later stage of fermentation in S. rochei.

Gamma-butyrolactone-dependent expression of the Streptomyces antibiotic regulatory protein gene srrY plays a central role in the regulatory cascade leading to lankacidin and lankamycin production in Streptomyces rochei.

Our previous studies revealed that the srrX and srrA genes carried on the large linear plasmid pSLA2-L constitute a gamma-butyrolactone-receptor system in Streptomyces rochei. Extensive transcriptional analysis has now showed that the Streptomyces antibiotic regulatory protein gene srrY, which is also carried on pSLA2-L, is a target of the receptor/repressor SrrA and plays a central role in lankacidin and lankamycin production. The srrY gene was expressed in a growth-dependent manner, slightly preceding antibiotic production. The expression of srrY was undetectable in the srrX mutant but was restored in the srrX srrA double mutant. In addition, SrrA was bound specifically to the promoter region of srrY, and this binding was prevented by the addition of the S. rochei gamma-butyrolactone fraction, while the W119A mutant receptor SrrA was kept bound even in the presence of S. rochei gamma-butyrolactone. Furthermore, the introduction of an intact srrY gene under the control of a foreign promoter into the srrX or srrA(W119A) mutant restored antibiotic production. All of these results confirmed the signaling pathway from srrX through srrA to srrY, leading to lankacidin and lankamycin production.

Transcription of the ehx enterohemolysin gene is positively regulated by GrlA, a global regulator encoded within the locus of enterocyte effacement in enterohemorrhagic Escherichia coli.

The pathogenicity island termed locus of enterocyte effacement (LEE) encodes a type 3 protein secretion system, whose function is required for full virulence of enterohemorrhagic Escherichia coli (EHEC). GrlR and GrlA are LEE-encoded negative and positive regulators, respectively, for controlling transcription of the ler gene, which encodes a central activator of LEE gene expression. We previously reported that the GrlR-GrlA regulatory system controls not only the LEE genes but also flagellar gene expression in EHEC (S. Iyoda et al., J. Bacteriol. 188:5682-5692, 2006). In order to further explore virulence-related genes under the control of the GrlR-GrlA regulatory system, we characterized a grlR-deleted EHEC O157 strain, which was found to have high and low levels of expression of LEE and flagellar genes, respectively. We report here that the grlR deletion significantly induced enterohemolysin (Ehx) activity of EHEC O157 on plates containing defibrinated sheep erythrocytes. Ehx levels were not induced in the grlR grlA double mutant strain but increased markedly by overexpression of GrlA even in the ler mutant, indicating that GrlA is responsible for this regulation. Ehx of the EHEC O157 Sakai strain is encoded by the ehxCABD genes, which are carried on the large plasmid pO157. The expression of ehxC fused with FLAG tag or a promoterless lacZ gene on pO157 was significantly induced under conditions in which GrlA was overproduced. These results together suggest that GrlA acts as a positive regulator for the ehx transcription in EHEC.

Single Circular Chromosome Identified from the Genome Sequence of the Vibrio cholerae O1 bv. El Tor Ogawa Strain V060002.

We report here the complete genome sequence of the Vibrio cholerae O1 bv. El Tor Ogawa strain V060002, isolated in 1997. The data demonstrate that this clinical strain has a single chromosome resulting from recombination of two prototypical chromosomes.