In Silico Prediction and Prioritisation of Novel Selective Antimicrobial Drug Targets in Escherichia Coli


 Treatment of infections caused by Escherichia coli and other Enterobacteriaceae often requires broad-spectrum antimicrobials, which cause perturbations of the gut microbiota (dysbiosis). Novel antimicrobial drugs interfering with pathogen-specific targets would minimize the risk of such dysbiosis. Here, we employed an in silico approach to identify essential proteins in E. coli, including pathogenic ST131, that are either absent or have low homology to humans and beneficial taxa of the gut microbiota. We identified 37 potential new targets with little or no homology to the proteomes seven taxa representative of the healthy gut microbiota. The suitability of these proteins as drug targets was further analysed through essentiality and conservation in the closely related pathogen Klebsiella pneumoniae. None of them are targets of commercially used antibiotics. Eighteen proteins are involved in four functionally connected essential biological processes (replication, chromosome segregation, cell division, and outer membrane biogenesis). Our results indicate that it may be possible to selectively interfere with essential biological processes in Enterobacteriaceae that are absent or mediated by unrelated proteins in beneficial bacterial taxa residing in the gut. The identified targets can be used to discover antimicrobial drugs that are effective against these opportunistic pathogens with a decreased potential of causing dysbiosis.


Introduction
Due to the worldwide increase in resistance observed among certain bacterial pathogens, there is a pressing need for novel antimicrobials. Most of the antimicrobial drugs approved for human use since the end of the antibiotic golden age in the 1960s belong to known antimicrobial classes and are primarily active against Gram-positive bacteria 1 . Thus, there is an urgent need for truly new antimicrobial compounds targeting Gram-negative bacteria. In 2017 the World Health Organization released a list of global priority antimicrobial-resistant (AMR) pathogens in order to guide research, discovery and development of new antibiotics 2 . The highest category ('Priority 1: Critical') comprises Acinetobacter baumannii, Pseudomonas aeruginosa and several Enterobacteriaceae, including Escherichia coli. The latter enteric species is one of the most frequent causes of bacterial infections. Speci c hyper virulent clones such as E. coli O25b sequence type (ST) 131 are responsible for a wide range of extra-intestinal diseases globally, ranging from urinary tract infection to septicaemia and meningitis 3 . Treatment of E. coli infections is often complicated by the high frequency of multidrug resistance. In particular, ST131 is a major contributor to the global spread of uoroquinolone resistance and extended-spectrum blactamase-mediated resistance to β-lactams, and is responsible for millions of multidrug-resistant infections each year 4 .
Oral antimicrobial therapy impacts the healthy gut microbiota by inducing a loss of bene cial microbes followed by expansion of opportunistic pathogenic bacteria, such as Enterobacteriaceae 5 . This phenomenon, generally referred to as dysbiosis, can in extreme cases lead to life-threatening secondary infections caused by Clostridioides di cile 6 , which are becoming increasingly common and di cult to treat. Certain antimicrobial-sensitive taxa residing in the healthy gut microbiota, e.g. Bacteroidetes and Lachnospiraceae have been shown to provide protection from C. di cile infections through colonisation resistance 5 Moreover, the gut-associated taxa Lactobacillus and Bi dobacterium both contain strains that are associated with bene cial effects on health and are used as probiotics in various food supplements, including Lactobacillus planetarum, L. paracasei, L. acidophilus, Bi dobacterium infantis, B. longum, and B. breve 7 .
Pathogen-targeted antimicrobial drugs with limited effect on bene cial organisms could potentially decrease the risk of dysbiosis and antibiotic-induced secondary infections. One approach to discover such drugs is the employment of target-based assays to identify compounds that selectively interfere with the viability of E. coli and other Enterobacteriaceae without affecting the healthy gut microbiota. This requires identi cation of targets that are speci c for this bacterial family. Here, we performed an in silico study to identify protein drug targets in E. coli that: i) are present in pathogenic E. coli ST131; ii) do not display signi cant homology to proteins in the host; iii) are absent or have low homology in selected members of the healthy gut microbiota. Furthermore, the selected targets were analysed for their conservation and essentiality in the closely related pathogen K. pneumoniae. Finally, the "druggability" of the selected targets was assessed based on subcellular localisation (SCL), availability of threedimensional structures and presence of known inhibitors.

Homology searches
The GenBank record for E. coli BW25113 (GenBank: CP009273.1) was downloaded, and the protein sequences from the 358 genes found to be essential by Goodall et al. 8 were extracted. Five genes were removed due to being labelled as pseudogenes (ttcC, yedN, ygeF, ygeN) or putative protein (yddL).
To establish presence of proteins in E. coli O25b:H4-ST131 (NCBI:txid941322), NCBI+ BLASTp was used to BLAST the 353 protein sequences against this organism using the non-redundant BLAST database. Percent alignment was calculated by dividing the length of the hit by the length of the query protein, extracted from the NCBI record. A dual cut-off was used, where hits E-value ≤ 10E -10 , or percent ID ≥ 70% and percent alignment ≥ 75% were excluded. These cut-offs were selected to be equal to, or more stringent than those used in previous in silico studies 52,53,56,57 . Any hits below the rst or above the second cut-off were removed, and the remaining were taken on to the next step.
To nd human analogues, the NCBI+ command line remote BLAST tool was used to BLAST the remaining protein sequences using rst the Entrez queries 'Homo sapiens [Organism]' against the nonredundant database, and the output was sorted using the same cut-offs as described above.

Presence in bene cial gut taxa
To establish presence in the simpli ed gut microbiota, remote BLASTp command line applications were used to search in speci c gut taxa using the Entrez queries ' The results were downloaded, and analysed using the same methodology and cut-offs as described above.
All hits sorted as above cut-off in the homology search against the simpli ed microbiota were collected and the number of hits for each protein manually evaluated. A table with the number of hits, together with the information for the highest scoring hit for each protein was generated. The proteins with less than one hits were manually inspected, and proteins with high scoring similarity were removed.
Presence and essentiality in K. pneumoniae To nd conservation in K. pneumoniae command line applications for BLASTp was used to BLAST remotely against 'Klebsiella pneumoniae subsp. pneumoniae KPNIH1 [Organism]' using the nonredundant database. The supplementary dataset generated by Ramage et al. 17 was downloaded and used to search for the potential target genes using gene names.

Subcellular localisation and structural information
The SCL for each protein was manually checked by querying the UniProt/SwissProt database, and retrieving the information found under 'Subcellular Localisation'.
Information about 3D structure for proteins in E. coli K-12 was manually retrieved through the UniProt/SwissProt entries for each protein individually. The PDB accession number, molecules in complex and resolution were recorded.

Dataavailability
The raw data generated during the current study are available from the corresponding author on reasonable request.

Results
Essential genes in the target pathogen Due to the lack of understanding of the essential genome in pathogenic strains such as ST131, the model strain BW25113 was used as a basis for our study. The predicted amino acid sequences of 353 out of the 358 essential genes identi ed by Goodall et al. 8 were retrieved (Figure 1). The remaining ve genes (ttcC, yddL, yedN, ygeF, ygeN), all labelled 'pseudogenes' or 'putative protein' and were not found in the Keio collection 9 , were excluded from further analysis. Their assignment as essential by Goodall et al. 8 may be thus an artefact of the methodology employed in the original study.
The sequences of the 353 proteins were compared to those found in E. coli O25b:H4-ST131 using a prede ned cut-off (E-value ≤ 10E -10 or ≥ 70% sequence identity, and ≥ 75% alignment length, see Materials and Methods). All of the 353 essential BW25113 proteins were associated with at least one hit in ST131, apart from YqeL (Supplementary Materials 1). However, 15 proteins were excluded as they scored below the cut-off threshold. Inspection of these sequences revealed that many of them were prophage-related or uncharacterized proteins. The presence of phages in a bacterial genome is expected to vary with the speci c strain history, and this may explain the observed difference between the laboratory strain BW25113 and the epidemic clonal lineage ST131. This left 337 essential and conserved E. coli proteins in the pipeline for further analyses.

Homology to proteins in mammalian hosts
The second step of the analysis aimed at removing E. coli targets homologous to the human proteome. A high degree of similarity between the pathogen's target and one or more proteins in the host proteome may result in off-target binding of a drug, leading to toxicity and unwanted side effects. The 337 selected essential proteins were therefore compared to the human proteome, leading to 186 proteins ful lling the same stringent cut-offs as above ( Figure 1, Supplementary Materials 2).
Homology to proteins in bene cial taxa of the gut microbiota The next step in the selection pipeline aimed to exclude proteins with high similarity to those found in representatives of the bene cial gut microbiota. Given the complexity and variability of the gut microbiome, we decided to focus on seven taxa containing species have previously been shown to have bene cial and protective effects on the host 5,6,10-16 : Faecalibacterium, Prevotella, Ruminococcus,Bacteroides, Lactobacillus, Lachnospiraceae and Bi dobacterium (Supplementary Materials 3-9). The 186 proteins were blasted against the abovementioned taxa using the same cut-off values as before ( Figure 1). As expected, this step was the most selective, leaving just 31 proteins to further analysis (Table 1) and removed all targets of commercially available antibiotics, including ParC (target of uoroquinolones), FtsI, MrdA (targets of b-lactams), parts of the 30S and 50S ribosome (targets of macrolides, aminoglycosides tetracyclines) and RNA polymerase (target of rifamycins).
Among the identi ed 31 proteins, only PheM and TrpL, each encoding a leader peptide in the Phe tRNA synthetase and Trp biosynthetic operon, were found to be missing completely in all taxa. No hits for YobI (a protein of unknown function) were found in any of the taxa apart from Faecalibacterium, where one single hit was found (E-value 5.1, 61.9 % alignment and 69.2% id). SafA (part of the low pH stress response) was found to be missing in Lachnospiraceae, Bi dobacterium and Faecalibacterium. Furthermore, WzyE (probable ECA polymerase), MreD (rod shape determining protein), LolA and LolB (both part of the lipoprotein transport pathway) lacked hits in Bi dobacterium, FtsL (a cell division protein) and MukF (involved in chromosome partition) in Bacteroides and YciS (lipopolysaccharide assembly protein A) in Faecalibacterium. All other proteins were associated with hits below the cut-off in all taxa.
Due to the high stringency applied in the above step, potentially valuable targets may have been missed in the selection process. Thus, a second analysis of the microbiota BLAST results was undertaken to nd proteins associated with only a few hits over cut-off. Six proteins (BamD, YfgZ, HolA, YrfF, LptD and ZipA) were each found to be associated with one hit only and had all been excluded based on E-value cut-off rather than sequence identity. Thus, these six proteins were included in further analyses, leading to 37 proteins as potential E. coli-selective targets (Table 1).
Target conservation in K. pneumoniae We evaluated presence and essentiality of the selected targets in K. pneumoniae KPNIH1, another global priority pathogen closely related to E. coli. The essentiality of the 37 proteins was checked against the library generated by Ramage et al. 17 , together with conservation of the amino acid sequence as above ( Figure 1, Supplementary Materials 10). Eighteen were found to be essential in both organisms, all displaying high sequence conservation, apart from ZipA and HipB. However, the majority of the targets not reported to be essential in K. pneumoniae ful lled the selection criteria, apart from HigA, IraM, SafA, YobI and TrpL (Table 1).

Biological function of selected targets
Of the 37 identi ed targets (Table 1), several were found to share or have similar biological functions ( Figure 2). One of the largest groups comprise of the proteins involved in outer membrane (OM) biogenesis and maintenance ( Figure 2). Here, BamD is directly associated with the OM, and is part of the b-barrel assembly machinery (BAM). LptA, LptD, LptE and LptF are all part of the lipopolysaccharide (LPS) transport (Lpt) machinery. LolA and LolB are found in the periplasmic space and the periplasmic side of the OM respectively, and belong to the lipoprotein transport machinery responsible for delivering OM lipoproteins to all three of the OM assembly machineries (LOL, BAM and LPT) 18 . SecE is part of the SecYEG protein translocation machinery responsible for transporting proteins into the periplasm 19 , and PssA is involved in phospholipid biosynthesis 20 . Furthermore, the inner membrane-protein YciS (also known as LapA, lipopolysaccharide assembly protein A) is part of a machinery responsible for envelope stress-response and regulation of LPS production 21 . Finally, although not associated with OM maintenance, TonB is part of the machinery responsible for actively importing iron across the OM in the cell 22 (Figure 2).
Another two functional groups comprise of the proteins responsible for DNA replication (HolA, HolD, PriB, DnaT and YgfZ) and cell division (MukB, MukE, MukF, FtsB, FtsL, FtsQ and ZipA) (Figure 2). DNA replication is a tightly controlled mechanism and DNA Polymerase III holoenzyme is the major replication complex in E. coli, where both HolA (d subunit) and HolD (y subunit) make up parts of the clamp loading complex 23 . DNA damage can cause this machinery to be stalled and disassemble on the chromosome, leading to replication failure. To re-start replication the cell must make use of the replication restart primosome, where both the PriB helicase and DnaT primase are found 24 . YgfZ has been shown to be part of the system regulating chromosomal replication 25 . MukBEF are unique to the g-proteobacteria and are involved in cell division, making up the only E. coli condensin for chromosome replication, segregation and organisation 26 . Further downstream in this process the transmembrane complex FtsBL is found 27 , together with FtsQ 28 and ZipA. In a related process, MreD is involved in determining cell shape 29 Among the proteins in the stress response category, CydX (Figure 2) is part of the CydAB cytochrome bd oxidase complex involved in aerobic respiration and maintaining the charge across the membrane used for synthesizing ATP 30 . IraM is a regulator of s S , the stationary phase sigma factor responsible for controlling expression of a plethora of genes involved in stress response 31 . Although the exact function of YobI has not yet been established, it has been shown to accumulate upon heat shock 32 .
Among the biosynthetic genes, WzyE has been implicated to be involved in assembly of the enterobacterial common antigen 33 , TrpL is involved in controlling tryptophan biosynthesis 34 and HemD is a uroporphyrinogen III synthase 35 (Figure 2).
HipB and HigA together make up the category of anti-toxins of the Type II Toxin-Antitoxin system, and work to counteract the effect of their cognate toxins 36 . As the sole members of their functional groups PheM is a target of transcriptional regulation ( Figure 2) and is responsible for attenuation of the phenylalanyl-tRNA synthetase 37 , while SafA is a two-component system connector 38 .
Finally, no information regarding biological function could be found for the three proteins YcaR, YrfF and YdhL ( Figure 2).

Target localisation
An essential requirement for to develop an e cient antimicrobial drug is target access. This is especially important in Gram-negative bacteria, where the double membrane structure acts as a permeability barrier, e ciently blocking many compounds from accessing intracellular targets. Subcellular localisation (SCL) was therefore considered to evaluate protein's druggability. Swiss-Prot, the manually annotated section of UniProtKB, was used to nd information on SCL for each of the 37 selected proteins ( Figure 2, Table 1). The target proteins were found to be located in either the Inner Membrane (IM), Outer Membrane, Cytoplasm, Nucleoid or Periplasm (Figure 2). Notably PssA was annotated as located in both the IM and the cytoplasm. For eleven proteins (PriB, HemD, DnaT, HolA, HolD, YdhL, HigA, HipB, PheM, TrpL and YobI), no SCL had been experimentally determined. Here, the four OM associated proteins (LptD, LptE, LolB and BamD) are promising potential targets, especially LptD, which contains extracellular domains.

Existence of known inhibitors
Next, the literature was searched for previously reported inhibitors of the selected targets. As expected, none of the targets presented in Table 1 are inhibited by commercially available antibiotics. Through analysis of scienti c literature we were able to identify inhibitors targeting a few of the listed targets but, to our knowledge, none has gone beyond laboratory studies: the ZipA/FtsZ interaction has been reported to be inhibited by certain antimicrobial compounds 39,40 ; the insect peptide Thanatin blocks LptA 41 ; compound IMB-881 blocks the interaction between LptA and LptC 42 ; JB-95 inhibits b-barrel proteins including LptD 43 ; MAC13243 inhibits LolA 44 ; BamD is inhibited by an inhibitory peptide 45 while the compound IMB-H4 has been shown to block BamA-BamD interaction 46 , and MukB is inhibited by the small molecules Michellamine B and NSC260594 47 . Finally, multiple inhibitory compounds targeting TonB have been identi ed [48][49][50] . Thanatin has been shown to possess antimicrobial activity against several Gram-negative bacteria beyond E. coli, including K. pneumoniae, Salmonella typhimurium and Enterobacter cloacae 41 . IMB-H4 was also able to inhibit growth in K. pneumoniae, P. aeruginosa and A. baumannii 46 . NSC176319 was found to be active against S. aureus and permeabilised P. aeruginosa and A. baumannii 47 . JB-95 was reported to have antimicrobial activity against A. baumannii, P. aeruginosa and Staphylococcus aureus 43 , MAC13243 has been shown to also be active against P. aeruginosa 44 and TonB inhibition has been shown to affect A. baumanii 49 . With the information provided in this study, some of these inhibitors may represent starting scaffolds for development into pathogen-speci c antibacterials. In addition, they might represent useful tools in validating future target-based assays.

Target structure
Structure-guided drug design is a powerful in silico approach that can rapidly screen millions of compounds for their ability to dock into a desired target, and identi ed hits can subsequently be tested in vitro. Thus, 3D structures at a high enough resolution represent an advantage for the targets identi ed in this study.
Information retrieved from the Protein Data Bank (PDB) 51 showed that 3D structures at a resolution of <3 Å existed for 18 proteins, >3 Å for 6 proteins and no structure could be found for 3 proteins, while YrfF was associated with a structure but no resolution information was reported in the database, and no structure had been reported for the remaining nine protein targets (Table 1).

Discussion
The originality of the present study lies in the identi cation of cellular targets that may lead to the discovery of innovative pathogen-selective antimicrobial drugs with limited effect on the healthy gut microbiota. Similar in silico studies have previously been conducted for verotoxigenic E. coli O157:H7, K. pneumoniae, Yersinia pseudotuberculosis and Enterobacteriaceae [52][53][54][55][56] . However, these studies were not designed to identify targets with low homology to the corresponding proteins in bene cial taxa residing in the intestinal tract or suffered from limitations related to the lack of a well-established essential genome for the target pathogen, or the comparisons to the human proteome.
We identi ed 37 potential drug targets selective for E. coli based on protein sequence homology. A large proportion of the identi ed proteins are functionally related between themselves. For example, MukBEF and FtsBL together with FtsQ and ZipA are all involved in regulation of the cell cycle; HolA, HolD and DnaT and PriB are part of two different complexes that are both part of the replication system in E. coli; PheM belongs to the separate, but related category of transcription regulation. OM maintenance was another large category, which is unsurprising as this structure is unique to Gram-negative bacteria while the majority of the bacteria used in this study as representatives of the healthy gut microbiota are Grampositive. Targets belonging to this category include proteins directly associated with the OM (LptD, LptE, BamD, LolB) as well as proteins found in various IM and periplasm related processes (LptA, LptF, LolA, SecE, YciS, TonB, MreD). The fact that multiple proteins from a single pathway were identi ed indicates that this is potentially a good cellular function to target. A number of proteins were found to be uniquely involved in various biosynthetic processes (HemD, PssA, TrpL and WzyE), stress response (CydX, IraM and YobI), or toxin-antitoxin systems (HigA and HipB). Proteins involved in two-competent system (SafA) or tRNA processing (YgfZ) were also found. Finally, four proteins without clear functions were identi ed (YcaR, YdhL, YgfZ and YrfF), indicating that there is more to discover regarding E. coli biology.
When searching for homologues in seven representative taxa of the healthy gut microbiota, only TrpL and PheM lacked hits in all these groups, indicating that these structures are unique to E. coli and possibly other opportunistic pathogenic bacteria residing in the intestinal tract. However, no info on SCL and 3D structure is available for either of these two proteins and only PheM is present and highly conserved in K. pneumoniae, although it is not proven to be essential in this species. Several proteins lacked hits in one or multiple taxa: YobI was only found in Faecalibacterium; SafA was missing Lachnospiraceae, Bi dobacterium and Faecalibacterium; no hits for WzyE, MreD, LolA, LolB and FtsL were found in Bi dobacterium; FtsL and MukF were missing in Bacteroides, and nally Faecalibacterium was lacking hits for YciS. These proteins may also be appropriate targets for developing pathogen-targeted antibiotics due to their absence in some gut bene cial bacteria. The remaining proteins were associated with hits below the cut-off, indicating that a similar protein may exist but that may be divergent enough that offtarget activity can be avoided.
The potential to target an infecting pathogen without affecting the bene cial microbiota makes E. coliselective antibiotics especially attractive. The clinical value of such antibiotics would be even higher if their spectrum covered other pathogenic bacterial species such. K. pneumoniae is a close relative to E. coli, and a major source of MDR infections in health care settings. The potential target proteins found in E. coli were therefore checked for sequence similarity and essentiality in K. pneumoniae. While the majority of the proteins were highly conserved in both species, only 18 out of the 37 targets were found to be essential in this pathogen. This could be due to disparity of information since the E. coli essential genome is well characterized, whereas the one in K. pneumoniae has been less studied. However, these 18 proteins are good candidates for developing pathogen-selective antibiotics that target both species.
An issue regarding essentiality in K. pneumoniae is its reliance on a relatively uncharacterised genome. The essentiality status is de ned by a single study, which may be affected by different parameters than the one used to identify the E. coli 'essentialome'. The lack of a well-characterised essential genome in K. pneumoniae leads to limitations in this selection step, as misclassi cation in terms of essentiality can occur and due to the scarcity of information and veri cation through consensus from multiple studies was not possible here. However, sequence conservation indicates that majority of proteins are highly conserved across the two species. Due to the close relativeness between the two species, investigations into the essentiality of the highly conserved but reported non-essential proteins may be worthwhile to further characterise their suitability as drug targets.
Due to the double-membrane structure found in Gram-negative bacteria, the accessibility of a potential drug target is essential. Proteins found in the OM are therefore considered to be targets with extra potential. Here, we identi ed LptD, LptE, LolB and BamD as OM associated proteins. Out of these proteins, LptD also has extracellular domains, indicating that it may be possible to nd an inhibitor that interferes with these portions and which would therefore not have to cross the OM. Interestingly, multiple of the OM proteins have already been targeted by various inhibitors, indicating that this is a viable strategy when developing novel antimicrobials. Notably, the four proteins listed above have an excellent 'druggability' potential since in addition to their optimal SCL, they are also essential in K. pneumoniae and have known 3D structures. Furthermore, all proteins were shown to be essential in K. pneumoniae and share a high degree of sequence similarity with this species, while they were found to only share low levels of sequence identity with the selected gut taxa used here. Here, LolB and LptE fell below the cut-offs used with alignment similarity ≤ 16%, and percent shared identity < 49%. Both LptD and BamD were recovered in the manual inspection of the proteins that were excluded due to falling above one of the cut-offs. However, both proteins were found to be excluded due to their E-value, and only be associated with one hit each: LptD in Bacteroides (12.5% alignment, 100% id and E-value 6,03E-62) and BamD in Lactobacillus (55.9% alignment, 24.8% id and E-value 6.06E-10). The selectivity of BamD and LptD can be further evaluated by future in vitro studies using known inhibitors that speci cally interfere with these proteins. 43,45,46 All in silico studies suffer from the drawback of cut-off criteria that may have little biological relevance. Stringent cut-off values potentially exclude valuable drug targets while loose criteria may result in an unmanageable list of targets. In the present study, while keeping stringent cut-offs throughout, we were able to identify 31 proteins present only in a selected number of bene cial gut microbes and worthy of further investigations. However, a manual analysis of the rejected sequences showed that six proteins could be recovered and that may be important targets for future drug development efforts.
Another issue related to in silico studies is that essentiality may differ between in vitro and in vivo conditions. The essential genome established by Goodall et al. 8 was characterized in rich media conditions, and therefore may miss genes essential for metabolism inside the host 8,57 . Certain biosynthetic pathways may be downregulated as the pathogen instead relies on the host to supply nutrients such as amino acids, vitamins and nucleobases 57 . However, targeting biosynthetic pathways involved in maintenance of the cell is likely to represent a target relevant in vitro as well as in vivo 58 .
In silico studies like this are a rst but essential step towards the discovery of novel pathogen-targeted antimicrobials. The results of our study provide a starting point towards the identi cation and development of novel speci c antimicrobials targeting E. coli. Future wet-lab studies are required to validate the presumptive selective targets identi ed by the study. High-throughput screens can be applied to nd inhibitors interfering with the speci c protein targets e.g. through knock-down strains with reduced expression of the target protein. The antimicrobial activity of the identi ed inhibitors could subsequently be evaluated on a comprehensive strain collection representative of the healthy gut microbiota or directly on faecal samples using a metagenomics approach in order to assess their selective toxicity towards E. coli and other pathogenic Enterobacteriaceae. Tables Table 1 The identi ed potential protein targets with gene name, PDB accession number, RCSB PDB structural information, Swissprot SCL, available inhibitors, sequence conservation and essential status in K. pneumoniae. N/A = Not available, N/D = Not determined.