In vivo evolution to hypermucoviscosity and ceftazidime/avibactam resistance in a liver abscess caused by Klebsiella pneumoniae sequence type 512.

Carbapenemase-producing Klebsiella pneumoniae represents a major public health issue globally. Isolates with resistance to the newest drugs, like ceftazidime/avibactam (CZA), are increasingly reported. In this study, we analyzed the evolution of KPC-3-producing sequence type (ST) 512 K. pneumoniae strains isolated at three different times (hospitalization days 45, 56, and 78) from the same patient, two of which were observed in a pericholecystic liver abscess. The three K. pneumoniae isolates (295Kp, 304Kp, and hmv-318Kp) from the same patient were subjected to antimicrobial susceptibility testing, whole-genome sequencing, sedimentation assay, biofilm measurement, serum resistance assay, macrophage phagocytosis, and adhesion assays. KPC-producing isolate hmv-318Kp exhibited carbapenem susceptibility, hypermucoviscous (hmv) colony phenotype and CZA resistance. Virulence markers of hypervirulent Klebsiella were absent. Two non-synonymous mutations were identified in the hmv-318Kp genome comparing with isogenic strains: a single-nucleotide polymorphism (SNP) occurred in the pKpQIL plasmid, changing blaKPC-3 in the blaKPC-31 gene variant, conferring CZA resistance; and a second SNP occurred in the wzc gene of the capsular biosynthesis cluster, encoding a tyrosine kinase, resulting in the F557S Wzc protein mutation. The Klebsiella pneumoniae strain exhibiting an hmv phenotype (hmv-Kp) phenotype has been previously associated with amino acid substitutions occurring in the Wzc tyrosin kinase protein. We observed in vivo evolution of the ST512 strain to CZA resistance and acquisition of hypermucoviscosity. The pathogenetic role of the detected Wzc substitution is not fully elucidated, but other Wzc mutations were previously reported in hmv K. pneumoniae. Wzc mutants may be more frequent than expected and an underreported cause of hypermucoviscosity in K. pneumoniae clinical isolates. IMPORTANCE: Here we describe the evolution of KPC-3-producing ST512 K. pneumoniae isolated at three different times from the same patient of which the last one, from a biliary abscess, showed CZA resistance by KPC-31 production and manifested hmv colony phenotype. Hypervirulent Klebsiella pneumoniae (hv-Kp) isolates are increasingly reported worldwide. Their hypervirulent traits are associated with the presence of rmpA/A2 genes and an hmv. In this study, we identified an hmv-Kp that lacked the rmpA-D cluster but showed an amino acid substitution in the Wzc tyrosin kinase protein, involved in the capsular biosynthesis. This hmv-Kp strain emerged in vivo and evolved resistance to ceftazidime/avibactam resistance in a liver abscess of a patient. Our findings suggest that wzc mutations may be underreported, making it challenging to distinguish hv-Kp from "classic" K. pneumoniae with an hmv phenotype.

Prevalence of multidrug-resistant hypervirulent Klebsiella pneumoniae without defined hypervirulent biomarkers in Anhui, China: a new dimension of hypervirulence.

Klebsiella pneumoniae is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes (rmpA and rmpA2) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the Galleria mellonella infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without rmpA and rmpA2 and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase (blaKPC - 2), ß-lactamase (blaOXA - 1, blaTEM - 1B), extended-spectrum ß-lactamase (ESBL) genes (blaCTX - M - 15, blaSHV - 106), outer membrane protein-coding genes (ompA), fimbriae encoding genes (ecpABCDER), and enterobactin coding genes (entAB, fepC). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci FEBNDAKP_03184 (c. 2084A > C, p. Asn695Thr), and EOFMAFIB_02276 (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the wzc of the capsule polysaccharide (cps) region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors.

Epidemiological characteristics of multidrug-resistant Acinetobacter baumannii ST369 in Anhui, China.

IMPORTANCE: Acinetobacter baumannii is a major health threat due to its antibiotic resistance and ability to cause nosocomial infections. Epidemiological studies indicated that the majority of globally prevalent ST369 clones originated from China, indicating a significant impact on public health in the country. In this study, we conducted whole-genome sequencing, comparative genomics, and Galleria mellonella infection model on eight A. baumannii ST369 isolates collected from a provincial hospital in China to comprehensively understand the organism. We identified two mutations (G540A and G667D) on the wzc gene that can affect bacterial virulence and viscosity. We confirmed their impact on resistance and virulence. We also investigated the potential involvement of AB46_0125 and AB152_03903 proteins in virulence. This finding provides a theoretical reference for further research on A. baumannii ST369 clinical isolates with similar mutations.

Urine-mediated suppression of Klebsiella pneumoniae mucoidy is counteracted by spontaneous Wzc variants altering capsule chain length.

Klebsiella pneumoniae is a hospital-associated pathogen primarily causing urinary tract infections (UTIs), pneumonia, and septicemia. Two challenging lineages include the hypervirulent strains, causing invasive community-acquired infections, and the carbapenem-resistant classical strains, most frequently isolated from UTIs. While hypervirulent strains are often characterized by a hypermucoid phenotype, classical strains usually present with low mucoidy. Since clinical UTI isolates tend to exhibit limited mucoidy, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract and select against mucoid isolates. We found that both hypervirulent K. pneumoniae and classical Klebsiella UTI isolates significantly suppressed mucoidy when cultured in urine without reducing capsule abundance. A genetic screen identified secondary mutations in the wzc tyrosine kinase that overcome urine-suppressed mucoidy. Over-expressing Wzc variants in trans was sufficient to boost mucoidy in both hypervirulent and classical Klebsiella UTI isolates. Wzc is a bacterial tyrosine kinase that regulates capsule polymerization and extrusion. Although some Wzc variants reduced Wzc phospho-status, urine did not alter Wzc phospho-status. Urine does, however, increase K. pneumoniae capsule chain length diversity and enhance cell-surface attachment. The identified Wzc variants counteract urine-mediated effects on capsule chain length and cell attachment. Combined, these data indicate that capsule chain length correlates with K. pneumoniae mucoidy and that this extracellular feature can be fine-tuned by spontaneous Wzc mutations, which alter host interactions. Spontaneous Wzc mutation represents a global mechanism that could fine-tune K. pneumoniae niche-specific fitness in both classical and hypervirulent isolates. IMPORTANCE Klebsiella pneumoniae is high-priority pathogen causing both hospital-associated infections, such as urinary tract infections, and community-acquired infections. Clinical isolates from community-acquired infection are often characterized by a tacky, hypermucoid phenotype, while urinary tract isolates are usually not mucoid. Historically, mucoidy was attributed to capsule overproduction; however, recent reports have demonstrated that K. pneumoniae capsule abundance and mucoidy are not always correlated. Here, we report that human urine suppresses K. pneumoniae mucoidy, diversifies capsule polysaccharide chain length, and increases cell surface association. Moreover, specific mutations in the capsule biosynthesis gene, wzc, are sufficient to overcome urine-mediated suppression of mucoidy. These Wzc variants cause constitutive production of more uniform capsular polysaccharide chains and increased release of capsule from the cell surface, even in urine. These data demonstrate that K. pneumoniae regulates capsule chain length and cell surface attachment in response host cues, which can alter bacteria-host interactions.

Hypermucoviscosity Regulator RmpD Interacts with Wzc and Controls Capsular Polysaccharide Chain Length.

Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.

Opposite evolution of pathogenicity driven by in vivo wzc and wcaJ mutations in ST11-KL64 carbapenem-resistant Klebsiella pneumoniae.

AIMS: To investigate the in vivo evolution of the mucoid-phenotype of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from the same patients and gain insights into diverse evolution and biology of these strains. METHODS: Whole genome sequencing and bioinformatic analysis were used to determine the mutation involved in the mucoid phenotype of ST11-KL64 CRKP. Gene knockout, bacterial morphology and capsular polysaccharides (CPS) extraction were used to verify the role of wzc and wcaJ in the mucoid phenotypes. Antimicrobial susceptibility, growth assay, biofilm formation, host cell adhesion and virulence assay were used to investigate the pleiotropic role of CPS changes in ST11-KL64 CRKP strains. RESULTS: Mutation of wzc S682N led to hypermucoid phenotype, which had negative impact on bacterial fitness and resulted in reduced biofilm formation and epithelial cell adhesion; while enhanced resistance to macrophage phagocytosis and virulence. Mutations of wcaJ gene led to non-mucoid phenotype with increased biofilm formation and epithelial cell adhesion, but reduced resistance of macrophage phagocytosis and virulence. Using virulence gene knockout, we demonstrated that CPS, rather than the pLVPK-like virulence plasmid, has a greater effect on mucoid phenotypic changes. CPS could be used as a surrogate marker of virulence in ST11-KL64 CRKP strains. CONCLUSIONS: ST11-KL64 CRKP strains sacrifice certain advantages to develop pathogenicity by changing CPS with two opposite in vivo evolution strategies.

Examination of the Impact of CYP3A4/5 on Drug-Drug Interaction between Schizandrol A/Schizandrol B and Tacrolimus (FK-506): A Physiologically Based Pharmacokinetic Modeling Approach.

Schizandrol A (SZA) and schizandrol B (SZB) are two active ingredients of Wuzhi capsule (WZC), a Chinese proprietary medicine commonly prescribed to alleviate tacrolimus (FK-506)-induced hepatoxicity in China. Due to their inhibitory effects on cytochrome P450 (CYP) 3A enzymes, SZA/SZB may display drug-drug interaction (DDI) with tacrolimus. To identify the extent of this DDI, the enzymes' inhibitory profiles, including a 50% inhibitory concentration (IC50) shift, reversible inhibition (RI) and time-dependent inhibition (TDI) were examined with pooled human-liver microsomes (HLMs) and CYP3A5-genotyped HLMs. Subsequently, the acquired parameters were integrated into a physiologically based pharmacokinetic (PBPK) model to quantify the interactions between the SZA/SZB and the tacrolimus. The metabolic studies indicated that the SZB displayed both RI and TDI on CYP3A4 and CYP3A5, while the SZA only exhibited TDI on CYP3A4 to a limited extent. Moreover, our PBPK model predicted that multiple doses of SZB would increase tacrolimus exposure by 26% and 57% in CYP3A5 expressers and non-expressers, respectively. Clearly, PBPK modeling has emerged as a powerful approach to examine herb-involved DDI, and special attention should be paid to the combined use of WZC and tacrolimus in clinical practice.

Evaluating NG-Test CARBA 5 Multiplex Immunochromatographic and Cepheid Xpert CARBA-R Assays among Carbapenem-Resistant Enterobacterales Isolates Associated with Bloodstream Infection.

Decreased susceptibility to carbapenems in Enterobacterales is an emerging concern. Conventional methods with short turnaround times are crucial for therapeutic decisions and infection control. In the current study, we used the Xpert CARBA-R (Cepheid, Sunnyvale, CA, USA) and the NG-Test CARBA 5 (NG Biotech, Guipry, France) assays for carbapenemase detection in 214 carbapenem-resistant Enterobacterales (CRE) blood isolates. We used the modified carbapenem inactivation method, conventional PCR, and sequencing to determine the production of five common carbapenemase families and their subtypes. We performed wzc-genotyping for all CR-Klebsiella pneumoniae (CRKP) and multilocus sequence typing for all carbapenemase-producing CRE isolates to reveal their genetic relatedness. The results showed a sensitivity of 99.8% and a specificity of 100% by the Xpert assay, and a sensitivity of 100% and a specificity of 99% by the NG-Test in detecting carbapenemases of 84 CRKP isolates with only one (VIM-1+IMP-8) failure in both tests. For CR-Escherichia coli, four carbapenemase-producing isolates were detected accurately for their subtypes. The two major clones of carbapenemase-producing CRKP isolates in Taiwan were ST11-K47 producing KPC-2 (n = 47) and ST11-K64 producing OXA-48-like (n = 9). Our results support the use of either test in routine laboratories for the rapid detection of common carbapenemases. Caution should be taken using the Xpert assay in areas with a high prevalence of CRE carrying blaIMP-8. IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) are emerging worldwide, causing nosocomial outbreaks and even community-acquired infections since their appearance 2 decades ago. Our previous national surveillance of CPE isolates in Taiwan identified five carbapenemase families (KPC, OXA, NDM, VIM, and IMP) with the KPC-2 and OXA-48-like types predominant. Timely detection and classification of carbapenemases in CPE may be a useful test to guide optimal therapy and infection control. Genetic detection methods using the Xpert CARBA-R assay and the immunochromatographic assay using the NG-Test CARBA 5 have been validated with the advantage of short turnaround time. Our study demonstrated that the NG and Xpert assays are convenient methods to accurately identify carbapenemases in carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Escherichia coli blood isolates. Detecting IMP variants remains challenging, and the results of Xpert CARBA-R assay should be carefully interpreted.

Phage Selective Pressure Reduces Virulence of Hypervirulent Klebsiella pneumoniae Through Mutation of the wzc Gene.

Hypervirulent Klebsiella pneumoniae (hvKp), one of the major community-acquired pathogens, can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment of K. pneumoniae, but the characteristics of the phage-resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two Podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. In vitro and in vivo experiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations of wzc and wcaJ genes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule. Meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. These findings contribute to a better understanding of bacteriophage therapy, which not only can kill bacteria directly but also can reduce the virulence of bacteria by phage screening.

Wuzhi capsule increased systemic exposure to methotrexate by inhibiting the expression of OAT1/3 and P-gp.

BACKGROUND: Methotrexate (MTX) is an important anticancer agent and immunosuppressant with a narrow therapeutic window. Wuzhi capsule (WZC) is an extract of Schisandra which is widely used to treat liver diseases. Co-administration of MTX and WZC is common in the clinical setting, but research on the interaction between WZC and MTX is limited. This study aimed to investigate the effects of WZC on the pharmacokinetics of MTX in rats and to explore the role of membrane transport proteins OAT1/3 and P-gp in the interaction of these drugs. METHODS: Plasma MTX concentration was detected by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS), and the messenger RNA (mRNA) and protein expression of OAT1/3 and P-gp was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting analyses, respectively. RESULTS: The study results revealed that co-administration of WZC decreased the CLz/F and Vz/F of MTX, increased the Cmax and area under the curve [(AUC)0-24 h] of MTX, and inhibited OAT1/3 expression in the kidney and P-gp expression in the small intestine. CONCLUSIONS: The findings suggested that there is a drug interaction between WZC and MTX and that OAT1/3 in the kidney and P-gp in the small intestine may be the main targets mediating the drug interaction, and attention should be paid when they are used in combination.

Investigation of the Impact of CYP3A5 Polymorphism on Drug-Drug Interaction between Tacrolimus and Schisantherin A/Schisandrin A Based on Physiologically-Based Pharmacokinetic Modeling.

Wuzhi capsule (WZC) is commonly prescribed with tacrolimus in China to ease drug-induced hepatotoxicity. Two abundant active ingredients, schisantherin A (STA) and schisandrin A (SIA) are known to inhibit CYP3A enzymes and increase tacrolimus's exposure. Our previous study has quantitatively demonstrated the contribution of STA and SIA to tacrolimus pharmacokinetics based on physiologically-based pharmacokinetic (PBPK) modeling. In the current work, we performed reversible inhibition (RI) and time-dependent inhibition (TDI) assays with CYP3A5 genotyped human liver microsomes (HLMs), and further integrated the acquired parameters into the PBPK model to predict the drug-drug interaction (DDI) in patients with different CYP3A5 alleles. The results indicated STA was a time-dependent and reversible inhibitor of CYP3A4 while only a reversible inhibitor of CYP3A5; SIA inhibited CYP3A4 and 3A5 in a time-dependent manner but also reversibly inhibited CYP3A5. The predicted fold-increases of tacrolimus exposure were 2.70 and 2.41, respectively, after the multidose simulations of STA. SIA also increased tacrolimus's exposure but to a smaller extent compared to STA. An optimized physiologically-based pharmacokinetic (PBPK) model integrated with CYP3A5 polymorphism was successfully established, providing more insights regarding the long-term DDI between tacrolimus and Wuzhi capsules in patients with different CYP3A5 genotypes.

Rational targeting of Wzb phosphatase and Wzc kinase interaction inhibits extracellular polysaccharides synthesis and biofilm formation in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic nosocomial pathogen, and responsible for high mortality and morbidity. Biofilm formation is one of the resistance determinants, where extracellular polysaccharide (EPS) is an essential component. EPS synthesis and its export is regulated by the bacterial Wza-Wzb-Wzc system. Wzc exhibits auto-phosphorylation protein tyrosine kinase activity, while Wzb is a protein tyrosine phosphatase. Wzb mediates dephosphorylation of Wzc. Dephosphorylated Wzc is required for the export of the EPS through porin Wza-Wzc complex. It shows that the interaction of Wzb with Wzc is critical for the export of EPS. Therefore, if the Wzb-Wzc interaction is inhibited, then it might hinder the EPS transport and diminish the biofilm formation. In this study, we have modelled the Wzb, and Wzc proteins and further validated using PSVS, ProSA, RAMPAGE, and PDBsum. The modelled proteins were used for protein-protein docking. The docked protein-protein complex was minimized by Schrodinger software using OPLS_2005 force field. The binding site of the minimized Wzb-Wzc complex was identified by Sitemap. The high throughput virtual screening identified Labetalol hydrochloride and 4-{1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino] propyl} phenol from FDA-approved drug library based on their interaction at the interface of Wzb-Wzc complex. The inhibitor-protein complex was further undergone molecular mechanics analysis using Generalized Born model and Solvent Accessibility (MMGBSA) to estimate the binding free energies. The lead was also used to generate the pharmacophore model and screening the molecule with antimicrobial scaffold. The identified lead was experimentally validated for its effect on EPS quantity and biofilm formation by A. baumannii. Wzb-Wzc interaction is essential for biofilm and EPS export; hence, the identified lead might be useful to regulate the biofilm formation by A. baumannii.

The role of the tyrosine kinase Wzc (Sll0923) and the phosphatase Wzb (Slr0328) in the production of extracellular polymeric substances (EPS) by Synechocystis PCC 6803.

Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ∆wzy (∆sll0737), ∆wzx (∆sll5049), ∆kpsM (∆slr2107), and ∆kpsM∆wzy (∆slr2107∆sll0737), strongly suggesting functional redundancy. In contrast, Δwzc (Δsll0923) and Δwzb (Δslr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.

Characterization of the wzc gene from Pantoea sp. strain PPE7 and its influence on extracellular polysaccharide production and virulence on Pleurotus eryngii.

To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test. The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane α-helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii.