The Wzi outer membrane protein mediates assembly of a tight capsular polysaccharide layer on the Acinetobacter baumannii cell surface

Abstract

Identification of novel therapeutic targets is required for developing alternate strategies to treat infections caused by the extensively drug-resistant bacterial pathogen, Acinetobacter baumannii. As capsular polysaccharide (CPS) is a prime virulence determinant required for evasion of host immune defenses, understanding the pathways for synthesis and assembly of this discrete cell-surface barrier is important. In this study, we assess cell-bound and cell-free CPS material from A. baumannii AB5075 wildtype and transposon library mutants and demonstrate that the Wzi outer membrane protein is required for the proper assembly of the CPS layer on the cell surface. Loss of Wzi resulted in an estimated 4.4-fold reduction in cell-associated CPS with a reciprocal increase in CPS material shed in the extracellular surrounds. Transmission electron microscopy revealed a disrupted CPS layer with sparse patches of CPS on the external face of the outer membrane when Wzi function was lost. However, this genotype did not have a significant effect on biofilm formation. Genetic analysis demonstrated that the wzi gene is ubiquitous in the species, though the nucleotide sequences were surprisingly diverse. Though divergence was not concomitant with variation at the CPS biosynthesis K locus, an association between wzi type and the first sugar of the CPS representing the base of the structure most likely to interact with Wzi was observed.

An update to the database for Acinetobacter baumannii capsular polysaccharide locus typing extends the extensive and diverse repertoire of genes found at and outside the K locus

“…Biosynthesis of A. baumannii CPS in the cytoplasm is known to begin with the transfer of a 'first' sugar to a lipid carrier in the inner membrane, and six possible first sugars transferred by one of six distinct initiating transferases (Itrs) belonging to one of two families (ItrA or ItrB) are known [ 55 ]. A seventh non-functional Itr type, ItrB2, has also been reported [ 11 ].…”

“…As wzx and wzy genes can be shared by K to wzx and wzy groups for future typing, a suffix was added to each wzx and wzy type (defined by the 85 % aa identity cutoff) indicating the name of the first KL a group was identified in. Finally, CPS assembly on the cell surface is mediated by a Wzi outer membrane protein encoded by a wzi gene located outside the K locus [55]. However, in rare cases, a second wzi type has been found at the K locus [54], and is referred to as wzi KL .…”

Genome-wide phage susceptibility analysis inAcinetobacter baumanniireveals capsule modulation strategies that determine phage infectivity

“…Transposon disruption of most of the 9 permissive KL3 genes led to higher fitness ( W ) during Loki exposure relative to untreated control (with 5 having non-FDR P < 0.05; Fig 3A). Disruption of wzi , an additional permissive gene outside the K locus that encodes the lectin required for efficient surface binding of exported capsular polysaccharide [51], also increased Tn-seq fitness during Loki challenge compared to control, albeit without passing statistical cut-offs (P = 0.15). Since a subset of the glycan building blocks encoded by the K locus are used for protein glycosylation by PglL [52], we also examined the pglL Tn-seq phenotypes.…”

Identification of distinct capsule types associated with Serratia marcescens infection isolates

“…coli , K . pneumoniae , and Acinetobacter baumannii [ 28 , 29 , 38 ]. In each case, when wzi is disrupted CPS no longer tightly associates with the cell surface and is found secreted in the supernatant.…”