Structural characterization of a Type B chloramphenicol acetyltransferase from the emerging pathogen Elizabethkingia anophelis NUHP1

Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.


Scientific Reports
| (2021) 11:9453 | https://doi.org/10.1038/s41598-021-88672-z www.nature.com/scientificreports/ the genus comprises three aerobic, non-motile rod-shaped Gram-negative species, including E. miricola, E. meningoseptica, and E. anophelis. Recent studies have identified potential new species that could be added to this genus, but further investigation is required 14 . Multiple Elizabethkingia strains have been isolated from a variety of environments, including human patients and mosquitoes 11 . Interestingly, E. miricola was isolated in 2003 from condensed water samples obtained from the space station Mir 15 . It has since been reported to cause pneumonia and lower respiratory tract infections, but the mechanism of transmission of the bacterium is unknown 16 . E. meningoseptica, previously identified as Chryseobacterium meningoseptica, is a hospital acquired pathogen that causes neonatal meningitis, pneumonia, and endocarditis 17,18 . E. anophelis is a relatively newly identified bacterium, and some investigations have suggested that it originated from the midgut of the Anopheles mosquito, Anopheles gambiae 19,20 ; however, it has also been isolated from Anopheles stephensi 21 . It causes similar infections as E. meningoseptica, which has made it challenging to clinically differentiate between these two organisms. As a result, E. anophelis infections have been underestimated due to their misclassification 22 . The E. anophelis bacterium responsible for the Singaporean outbreak was isolated from a human patient and designated as the NUHP1 strain. NUHP1 is differentiated from other E. anophelis strains because it acquired an ICEEa1 integrative conjugative element (ICE) within its mutY gene. ICE is a mobile genetic element that integrates into the host chromosome, replicates, excises, and forms a plasmid in order to be transferred to other bacterial cells via horizontal conjugation. These mobile genetic elements are used by bacteria to enhance survival in diverse environments and often contain antibiotic resistance genes. Within the Flavobacteriaceae family, only E. anophelis and R. anatipestifer have been shown to contain ICE 23,24 . Notably, only strains of E. anophelis with ICE have caused outbreaks. While ICE contain antibiotic resistance genes, additional antibiotic resistance genes are found outside these regions on the chromosome and contribute to bacterial survival. Within the E. anophelis NUHP1 genome, 14 antibiotic resistance genes have been identified, including ones required for resistance to aminoglycosides, beta-lactams, macrolides, tetracycline, trimethoprim, and chloramphenicol (Cm) 25 . One example of an antibiotic resistance gene that is conserved in all Elizabethkingia strains is the chloramphenicol acetyltransferase (CAT) gene 26 . To our knowledge this gene has not been described as being located in an ICE.
Since the E. anophelis NUHP1 strain has caused significant outbreaks in recent years, the Seattle Center for Structural Genomics of Infectious Diseases selected antibiotic resistance proteins from this pathogen for structural determination. One of these proteins is CatB, which is predicted to be a chloramphenicol acetyltransferase. Homologs of this protein are important for bacterial Cm antibiotic resistance because they acetylate the 3′-hydroxyl group of Cm (Fig. 1A). This covalent chemical modification prevents Cm from inhibiting bacterial protein synthesis in ribosomes. CAT enzymes are classified based on their origin and their sequence and structural homology. They have been categorized into three predominant types: Type A, Type B and Type C 27 . These are also sometimes referred to as CatA, CatB, and CatC. A defining characteristic between these CATs is that Type B and C display a lower apparent affinity for Cm than Type A 27 . Additionally, Type A CATs form a distinct structural group compared to Type B and C. All three types of proteins are trimers, but Type B and C adopt a hexapeptide repeat structural fold, which is not found in Type A (Fig. 1B-D). Since the E. anophelis catB gene is conserved across all Elizabethkingia strains and CatB proteins are critical for Cm resistance in important

Materials and methods
The E. anophelis NUHP1 catB gene (UniProt ID: A0A077EJ45) was cloned, expressed, and purified as described 30 .
In preparation for crystallography, E. anophelis CAT was concentrated to 21 mg/ml in 25 mM HEPES/NaOH, pH = 7.0, 500 mM NaCl, 5% glycerol, 2 mM DTT, 0.025% NaN 3 (SSGCID batch ID ElanA.01572.a.B1.PW38419). The protein was further diluted to 19.5 mg/ml upon the addition of 5 mM MgCl 2 and 2.5 mM acetyl-CoA and 2.5 mM chloramphenicol and incubated for 10 min at 287 K. Crystals were then grown at 287 K by sitting drop vapor diffusion in XJR trays. A volume of 0.4 μl of protein/ligand complex was mixed with 0.4 μl of JCSG + , well A11 reservoir solution (Rigaku Reagents, Bainbridge Island, WA): 50% (v/v) MPD, 0.1 M Tris base/HCl, pH = 8.5, 0.2 M ammonium phosphate monobasic. The reservoir volume was 80 μl. Crystals were harvested and flash-frozen in liquid nitrogen. Data were collected at 100 K on a Rayonix MX-300 mm CCD detector at a wavelength of 0.97872 Å on beamline 21-ID-F at Life Sciences Collaborative Access Team (LS-CAT) at the Advanced Photon Source (APS, Argonne, IL). Data were reduced with the XDS/XSCALE package 31 . The structure was solved using molecular replacement with MorDA 32 and PDB ID: 1XAT 29 as a starting model. Iterative rounds of manual model building and automated refinement were carried out using Coot 33 and Phenix 34 . The quality of the structure was checked by Molprobity 35 , and the final structure was deposited into the Protein Data Bank (PDB) using the code 6MFK. However, the protein was produced with a non-cleavable N-terminal polyhistidine tag (MAHHHHHH) and two C-terminal histidine residues of the tag were partially ordered. The backbone atoms of these His residues and residues 1 and 2 of the protein were ordered. Both the backbone and the side chains of the rest of the residues (3-208) were ordered and included in the model.

Structural analysis of E. anophelis NUHP1 CAT protein.
To characterize the type of CAT E. anophelis NUHP1 harbors, we determined the structure of the protein encoded by the catB gene using X-ray crystallography ( Table 1). The electron density map allowed all residues of the protein (1-208) to be modeled. A single protein monomer was present in the asymmetric unit of the crystal with the topology shown in Fig. 2A. The monomer contained two main domains: (1) a left-handed β-helix core (15 β-strands spanning residues 12-163) forms a prism at the center of the monomer and contains two extended loop regions (44-56 and 72-110), and (2) a C-terminal α-helical domain comprised of three α-helices (spanning residues 164-208) (Fig. 2B). To determine whether the E. anophelis CAT was likely to form a multimer, we examined the crystal structure by expanding the asymmetric unit and investigated whether large interfaces may be present using Proteins, Interfaces, Structures and Assemblies (PISA) 24 . The only biological assembly predicted from PISA for the E. anophelis CAT enzyme was a trimer, which is identical to all other structurally characterized CAT enzymes. (Fig. 2C). To characterize the interfacial residues that are important for this biological assembly, and to reveal the conservation of these residues in other CAT enzymes, we compared these interfaces with other structures of CATs . In our E. anophelis CAT structure, we found one interface mediating the trimer formation (Supplementary Figure 1). This interface is mediated through a large number of polar and non-polar interactions. In total, the interface forms 21 hydrogen bonds, two salt bridges (Supplementary Tables 1 and 2 Table 3). Next, we compared this biological interface with related CAT enzymes containing acetyl-CoA (PDB ID: 6U9C 27 ), and Cm (PDB ID: 2XAT 29 ). We found very similar interfaces in these ligand and cofactor bound structures, with CAT bound to acetyl-CoA exhibiting 23 hydrogen bonds, 2 salt bridges, and 1612 Å 2 ASA, and CAT bound to Cm exhibiting 16 hydrogen bonds, 2 salt bridges, 1391 Å 2 ASA (Supplementary Tables 1 and 3).
The E. anophelis protein exhibits greatest structural homology with Type B CATs. To determine which type of CAT the E. anophelis protein most closely resembles, we performed a pairwise sequence alignment and a structural comparison with representative sequences and structures of different categories of www.nature.com/scientificreports/ CATs. To begin, we selected sequences of three different types of CAT proteins that had been structurally characterized: Type A from E. coli (catI; UniProt ID: P62577), Type B from P. aeruginosa (catB7; UniProt ID: P26841), and Type C from A. fischeri (UniProt ID: Q5DZD6). We performed a multiple sequence alignment with the E. anophelis CAT and these three proteins and found the E. anophelis CAT shared 17%, 62%, and 54% identity with Type A, Type B, and Type C CATs, respectively (Supplementary Figure 2). The insertion typically found in Type C CATs was not present in the E. anophelis protein. Therefore, the E. anophelis protein most closely resembled Type B CAT protein sequences. To further explore whether the E. anophelis protein structure also resembled Type B CATs, we performed a structural comparison of this protein with other types of CATs that had been structurally characterized. We specifically compared the E. anophelis 6MFK structure with the E. coli 3U9F 28 , P. aeruginosa 2XAT 29 , and A. fischeri 5UX9 27 structures. The Type A protein from E. coli adopts a completely different fold than the Type B and C proteins from P. aeruginosa and A. fischeri, respectively; Type B and C CATs adopt a hexapeptide repeat fold (Fig. 1). The E. anophelis 6MFK structure superimposed well with the Type B 2XAT structure 29 (rmsd 0.6 Å) and Type C 5UX9 27 structure (rmsd 1.1 Å). Therefore, the results of the sequence and structural comparisons indicate the E. anophelis protein is most likely a Type B CAT.
Putative active site residues of the E. anophelis CAT protein and other Type B CATs are conserved. Next, we examined the putative active site residues of the E. anophelis CAT protein and compared them to previously determined Type B CAT structures in complex with substrates or substrate analogs. CAT proteins have two binding sites for each substrate (Cm and AcCoA), which are located at the interface between monomers of the trimer. To determine which residues are located in both sites of the E. anophelis CAT, we superimposed its structure with the P. aeruginosa catB7 (PDB ID: 2XAT 29 ; rmsd 0.6 Å) structure in complex with Cm and desulfo-coenzyme A and the V. cholerae catB9 (PDB ID: 6U9C 27 ; rmsd 0.6 Å) structure in complex with AcCoA. The trimeric E. anophelis CAT structure was built based on the 6U9C 27 structure and ligands from the 2XAT 29 and 6U9C 27 structures were modeled into the trimer to compare putative active site residues. The residues that could potentially hydrogen bond with Cm in the binding site of the E. anophelis structure include Pro8, Gly11, Tyr30, and Ser32 from one monomer and His79 from a second monomer (Fig. 3). These residues are all identical in the CAT enzyme from P. aeruginosa (PDB ID: 2XAT 29 ) (Fig. 3).Residues critical for mediating H-bonding interactions with AcCoA in the 6U9C 27 structure included Ser137 and Lys160 from the first monomer and Thr142 in the second monomer. The corresponding residues in the 6MFK structure are Ser139, Lys162, Thr144 (Fig. 4).
Once we identified the key putative active site residues in the E. anophelis CAT structure, we examined the conservation of these residues across sequences of other Flavobacteriaceae pathogens (Fig. 5). To begin, we aligned the E. anophelis CAT protein sequence to others identified by BLASTp against the genomes of the following pathogens: E. miricola, E. meningoseptica, Chryseobacterium sp., and R. anatipestifer. We found the E. anophelis CAT protein shares 95% sequence identity with E. miricola, 86% identity with E. meningoseptica, 83% identity with Chryseobacterium sp., and 81% identity with R. anatipestifer Type B CAT proteins. Additionally, the putative active site residues for both Cm and AcCoA sites identified in the E. anophelis CAT protein were highly conserved across all of the CAT sequences from these pathogens.

E. anophelis CatB is similar to clinically validated Cm resistance Proteins.
To determine whether the E. anophelis CAT protein was similar to known Cm resistance proteins in different pathogens, we searched the Comprehensive Antibiotic Resistance Database (CARD) 36 using the E. anophelis CAT protein sequence. The search yielded nine proteins from a variety of bacterial pathogens with validated Cm resistance profiles (Table 2; Fig. 5). All of these proteins were Type B CATs and they shared between 62-73% sequence identity with the E. anophelis CAT. When we compared their active site residues with the E. anophelis CAT protein, we saw all Cm binding site residues were conserved. The only variation in residue conservation was in the AcCoA binding site. The majority of the residue substitutions were of similar chemical properties (e.g. substituting Val for Ile). However, one exception to this pattern was observed: the CatB7 protein from P. aeruginosa had a Gly residue in the corresponding location of Lys145 in the E. anophelis protein. Based on these results, it is highly likely that the E. anophelis CAT and its homologs in other Flavobacteriaceae pathogens could also catalyze the acetylation of Cm and confer resistance to this antibiotic.

Discussion
E. anophelis has been discovered in diverse environmental and host associations, including its presence in soil and water and its isolation from the African and Asian malaria vector mosquitoes Anopheles gambiae and Anopheles stephensi. Currently it is unclear whether the mosquito itself can be a source of E. anophelis transmission to humans, or if other mechanisms occur, including potential reservoirs within hospitals such as sink basins and water faucets 37 . In a study examining different E. anophelis strains, those isolated from the midgut of a mosquito contained genes that encode a xylose isomerase and xylulose kinase, which human isolates lacked. This suggests that different environments may have specific requirements for sugar metabolism, and clinical and environmental strains of E. anophelis differ from one another 19 . Some other Flavobacteriaceae pathogens have been shown to be transmitted in the air, on surfaces, in water, and by ingesting contaminated food 38 . They can also be part of the normal flora of the throat of some duck species 39 .
E. anophelis harbors a variety of antibiotic resistance genes along with several multidrug efflux pumps, which are speculated to contribute to the shape and stabilization of the microbial community within the gut of the mosquito 40 . In addition to antibiotic resistance genes, multiple virulence factors contribute to Elizabethkingia species survival and persistence. For example, E. meningoseptica contains various virulence factors, including those that contribute to proteolysis, iron uptake and transport, biofilms, and capsule formation. Bacterial persistence Scientific Reports | (2021) 11:9453 | https://doi.org/10.1038/s41598-021-88672-z www.nature.com/scientificreports/ on surfaces, including cellular adhesion and medical devices, is facilitated by these virulence factors via biofilm, capsule and sometimes curli formation. Biofilms have been shown to be critical for E. meningoseptica infections and also allow these bacteria to resist surface disinfection 41 . Cm has anti-biofilm properties and is effective at killing a variety of bacteria and preventing colony growth 42 ; however, this has not been investigated for Elizabethkingia species. Cm is not typically used to treat internal bacterial infections due to serious side-effects and toxicity, but it is more widely used as a topical treatment or for some ocular infections 43 . Because Cm is not widely prescribed, some current bacterial pathogens are still susceptible to this antibiotic. Therefore, renewed interest in salvaging or repurposing this drug to treat bacterial infections is also being considered as a viable path forward 44 . While it is unlikely this drug would be used to treat an E. anophelis infection in the current pharmaceutical climate, it is possible Cm would be considered in the future as our arsenal of effective antibiotics becomes reduced. Moreover, Cm is an effective drug that can efficiently pass the blood-brain barrier 45 , which is important for treating meningitis infections like those associated with E. anophelis or E. meningoseptica. Thus, knowing the E. anophelis NUHP1 bacterium contains a Cm resistance gene may be important for future therapeutic considerations. www.nature.com/scientificreports/ Our analysis of the E. anophelis CAT protein has shown it adopts a similar structure and retains conserved putative active site residues as clinically validated Type B CAT proteins in other bacterial pathogens. While we recognize in vitro enzyme kinetics assays are needed to ensure the E. anophelis enzyme can actually acetylate Cm, the near 100% conservation of putative active site residues compared to other Type B CATs that have been kinetically characterized suggests they are functionally similar. Even though Type B CATs acetylate Cm and are important for Cm resistance, their native functions remain elusive. Several other hexapeptide repeat acetyltransferases have been explored and have been shown to acylate different sugars or cell wall polysaccharides 46,47 . However, these characterized enzymes are still divergent in sequence and structure from the Type B CATs. Therefore, further investigation into the native function of the Type B CAT enzymes is warranted, especially since these proteins are widely found in opportunistic and colonizing pathogens. It would be particularly interesting to explore the native function of the CatB protein from E. anophelis since it has an extraordinarily flexible biological capacity to live in a wide array of environments and at times can colonize humans and cause disease.
Despite our lack of knowledge of the native function of the E. anophelis CAT protein, the gene is conserved in all identified environmental and nosocomial E. anophelis strains 48 . Our analysis of the Type B CAT protein sequence across Elizabethkinigia species showed most of the protein sequence is conserved; however, there were some key amino acid changes between the proteins from different species. Due to the difficulty in accurately differentiating Elizabethkingia species during genome sequencing 22 , this gene may be used as a tool to improve species identification of isolates and propagation of different strains around the world.