Saturday, October 29, 2011

Bacterial O-antigens, capsules, and cell-surface polysaccharides: not just all-sugar

You probably heard of Escherichia coli O104:H4, which caused a devastating outbreak of an enterohemorrhagic disease in many  European countries this year. Did you ever wonder what the O and H in the name represent? In the pre-genome sequence era, enterobacteria were usually distinguished based on the type of their polymorphic surface antigens by a process called serotyping. In this, antibodies that specifically recognized a distinct type of surface antigen were used to identify the bacterial serotype. This was an extraordinarily successful tool in epidemiological studies. In  enterobacteria, the polymorphic surface molecules are typically a surface lipopolysaccharide (O-antigen), flagellar proteins (H antigen) and/or the capsular polysaccharide (K-antigen). Thus the O104:H4 in the E.coli strain name refers to the type numbers of the O and H antigens respectively. E. coli has about 700 serotypes combined from some 180 O-antigens, 70 K-antigens and 54 H-antigens. Salmonella has about 2500 serotypes! Below we highlight a new twist to the O-antigen structure that we recently uncovered in our study on peptide ligases.

Let us study the Lipopolysaccharide (LPS), of which the O-antigen is a component, in some more detail (see figure below).The LPS is comprised of four components. 1) Lipid Aa lipid anchor that forms the outer monolayer of the outer membrane and anchors the LPS, 2) an inner core composed of characteristic sugars such as Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) and a heptose,   3) an outer core typically containing hexose sugars, and finally  (4) the O-antigen repeats that  exhibit variations in the type and arrangement of the sugar residues within the O-unit of LPS (see figure below). Some O-antigens  have repeats of 3-5 sugar units, others are branched with 4-6 sugar units. Also present are unusual sugars only seen in these surface antigens.The number of such repeats also greatly vary (See the O-antigen database). Estimates suggest that there are about a million LPS molecules sticking out from the outer membrane per E. coli cell. The variations are a means for the bacterium to escape the surveillance of the host immune system  and function as a virulence factor. Additionally, the antigens might vary to avoid bacteriophages that target the O-antigen for attaching and invading the bacterial cell. The genes involved in the biosynthesis of the O-antigen are present in a large gene cluster and not unexpectedly show great variations between various O-antigen types. Many of these are involved in the biosynthesis and export of the sugar units in the LPS. 

                                              O-antigen structure (from Raetz and Whitfield)
In a recent study, we noticed a somewhat unexpected presence in these gene neighborhoods-- peptide ligases. The proteins encoded by the E.coli/Shigella wfdG and wfdR  O-antigen cluster genes (incorrectly labeled as glycosyltransferases) are members of the ATP-grasp superfamily of peptide ligases. Members of this family are present widely across bacteria, e.g. firmicutes, actinobacteria, proteobacteria, spirochaetes, bacteroidetes, fusobacteria and cyanobacteria. Interestingly, they are also present in the capsular biosynthesis locus of Streptococcus pneumoniae (e.g. wcyv).  In general, this family of peptide ligases are combined with genes that encode proteins involved in biosynthesis of cell surface polysaccharides. In some instances members of this family are fused to other domains such as glycosyltransferases and the capsular biosynthesis-type PP2A-fold phosphatases. Often these neighborhoods encode multiple paralogous copies of ATP-grasps (access the operons here).  Pioneering studies in Proteus and Providencia (e.g. Kocharova et al. and Kondakova et al) have shown that sugars of the cell surface O-antigen are further aminoacylated by D- and L-aspartic acid residues. Given the presence of  ATP-grasp genes in these operons, we predict that they would catalyze the ligation of amino acids to sugar moieties in these polymers, as observed in these studies. 

One other cell surface polysaccharide with known sugar-amino acid conjugates is  teichuronopeptide, a highly acidic copolymer of glucuronic acid and amino acids such as glutamate that contributes to alkaliphily of organisms such as Bacillus halodurans. Experimental studies by Aono had implicated the TupA gene in the biosynthesis of this product but the mode of action was not understood until we unified TupA to the same family of ATP-grasps (TupA-like) present in the O-antigen and capsule biosynthesis loci. We predict that this is the ligase required for synthesis of the polyglutamate portion of the teichuronopeptide. The Teichuronopeptide synthesis locus additionally contains three paralogous ATP-grasp genes (see operons here). A comparable combination of gene neighborhoods is also seen in alkali resistant bacteria such as Dethiobacter alkaliphilus and Oceanobacillus, and the polycyclic aromatic hydrocarbon degrading Mycobacterium sp. JLS.  This suggests that the teichuronopeptide-like polymer might have been an important solution to the problem of high alkaline or salt conditions. The lateral transfer of this neighborhod might have been important in the emergence of alkali resistance in various distantly related bacteria. 
Teichuronopeptide unit
The wide phyletic distribution of this ATP-grasp-centered and related operons suggests that sugar/sugar acid and amino acid conjugates are a common feature of the capsules and other distinctive cell surface polymers of a large number of bacteria. The presence of up to four ATP-grasp genes in some of these operons suggests peptide chains with complexity comparable to the peptide linkages in peptidoglycan might be present in some of these polymers. This throws an exciting twist to the composition of the cell surface polysaccharides of bacteria. The nature and type of amino acids in these various species would definitely be of great interest and importance to bacteriology and epidemiology. You can access our paper here and browse the extensive supplement here.