performed structural analyses of O-antigens. recognized as urgent threat level pathogens requiring immediate actions (CDC: Antibiotic Resistance Threats, 2013). Among CRE, strains are by far the most common isolates. With acquisition of the recently described plasmid mediated colistin resistance1, pan-resistant strains have emerged2. Given the dwindling development pipeline of novel antibiotics with Gram-negative coverage, alternative therapeutic options against extremely drug resistant Gram-negative Momordin Ic bacteria are needed. Passive immunization using pathogen-specific monoclonal antibodies (mAbs) is an approach envisioned to be in good accordance with proposed antibiotic stewardship efforts. Application of mAbs does not induce antibiotic resistance, preserves the normal microbiota, and hence may also be used for prophylaxis in high-risk patient populations. LPS O-antigens show limited variability and consequently were proposed as viable targets for immunization3C5. Former and recent epidemiology data suggest that 3 to 4 4 serogroups constitute more than 80% of all clinical isolates4C6. Efficacy of mAbs against strains expressing the most common galactan-based O-specific polysaccharides (O-PS) (O1 and O2) was previously shown7, 8. The remaining common serogroups, O3 and O5, express O-PS chains made up of mannan (i.e. homopolymer of mannose) structures that are identical to the O9 and O8 O-PS structures, respectively9, 10. The O3 and O5 repeating units (RU) differ in their number of mannoses, anomeric configurations and/or their intra-mannose linkages11. The non-reducing ends of both O3 and O5 side-chains are terminated with different structures, methylphosphate (MeP) and methyl (Me) Momordin Ic groups, respectively11, 12. O-PS termination is coupled to chain polymerization in the WbdA-WbdD complex, which Momordin Ic is anchored in the inner face of the inner membrane13, 14. WbdA is a multi-domain mannosyltransferase15; different domains of this enzyme catalyse the addition of mannose molecules at different linkages to the O-antigen polymer. A single point mutation in one of the mannosyltransferase domains of WbdA in O9 results in the reduction of 5 mannose molecules to 4 within the RU16. The WbdD enzymes are responsible for the serogroup-specific termination17. O-PS termination is the signal for ABC-mediated transport across the inner membrane18 followed by the ligation of O-antigens to the preformed lipid A-core templates. In this study, we describe novel O3 subtypes that express O-antigen RUs comprising 3 or 4 4 mannoses in contrast to the 5 mannoses originally described. The novel variants carry mutations in the mannosyltransferase domains of WbdA, which can explain the observed structural differences. Moreover, in support of the clinical significance, we have selected two classes of mAbs that are either specific to certain subtypes or cross-reactive to all O3 variants and therefore can be considered for diagnostic (seroepidemiology) or therapeutic purposes, respectively. Results Development of mAbs with different specificity to O3 strains Mouse hybridomas were generated using splenocytes from BALB/c mice immunized with prototype strain PCM-11 (O3:K11: this strain, in fact, is proven in this study to express an O3a type O-antigen). Reactive clones specific to the purified and biotinylated O-PS of a non-encapsulated mutant, 5505(O3:K-) were selected by screening hybridoma supernatants by ELISA. Subsequently, the purified mAbs were tested by immunoblotting with a panel of LPS samples obtained from different O3 clinical isolates identified by O3-specific PCR using Momordin Ic primers specific to the genes (Fig.?1). Interestingly, two classes of mAbs were found: i) those reacting with LPS from all O3 strains (exemplified by mAb 2F8, Fig.?1a) and ii) those showing different binding intensities (from strong to negligible binding) to LPS from various O3 Mouse Monoclonal to His tag strains (as shown with mAb 1G6, Fig.?1b). None of these mAbs stained any of the non-O3 LPS molecules, such as O1, O2, and O5 (Fig.?1, lanes 1, 2, and 3, respectively) indicating lack of poly-reactivity. Open in a separate window Figure 1 Immunoblot (a,b) and ProQ staining (c) of LPS samples. LPS was Momordin Ic purified from different O3 (lanes 4C9) and unrelated serotype (lanes 1C3) strains of O3 strains with monoclonal antibodies. O3-specific or a control murine mAb was incubated.