New Results

Expanding the toolbox: Novel class IIb microcins show activity against gram-negative ESKAPE and plant pathogens

Benedikt M. Mortzfeld, Shakti K. Bhattarai, Vanni Bucci
doi: https://doi.org/10.1101/2023.12.05.570296

Abstract

Interspecies interactions involving direct competition via bacteriocin production play a vital role in shaping ecological dynamics within microbial ecosystems. For instance, the ribosomally-produced siderophore bacteriocins, known as class IIb microcins, have been observed to affect the colonization of pathogenic Enterobacteriaceae species within hosts. Notably, to date, only five of these antimicrobials have been identified and all were derived from specific Escherichia coli and Klebsiella pneumoniae strains. We hypothesized that class IIb microcin production extends beyond these specific compounds and organisms. By employing an informatics-driven approach, screening bacterial genomes in publicly accessible databases, we have discovered a total of twelve previously unknown class IIb microcins. Our investigation unveiled that these microcins are harbored within a diverse array of Enterobacteriaceae species, encompassing phytopathogens and environmental isolates. We introduce three novel clades of microcins (MccW, MccX, and MccZ), while also identifying eight new variants of the five previously known ones. To validate their antimicrobial potential, we heterologously expressed these microcins, along with their immunity peptides, in E. coli and unequivocally demonstrated their efficacy against a variety of bacterial isolates, including plant pathogens like Gibbsiella species and Rahnella victoriana. Remarkably, two of these newly discovered class IIb microcins exhibit activity against gram-negative ESKAPE pathogens, such as Acinetobacter baumannii or Pseudomonas aeruginosa providing the first evidence that class IIb microcins can target bacteria outside of the Enterobacteriaceae family. Our findings hold significant promise for the development of innovative live biotherapeutic products tailored to combat these resilient bacteria and underscore the notion that class IIb microcins are more prevalent and more broad-spectrum in the natural microbial world than previously recognized.

Introduction

A large body of theoretical and experimental work has shown that dynamics of microbiomes are shaped by the network of interbacterial interactions(1–4). These cooperative and competitive interactions are often achieved via the secretion of cross-feeding metabolites(5), antimicrobial peptides(6) and bacterially produced small molecules(7,8) and are crucial to ecological properties including stability and ability to respond to external perturbations(9,10). Among the competitive interactions, bacteriocin production is proposed to be a prominent mediator of microbiomes dynamics(11) and, specifically, several reports including ours have shown that a bacteriocins subclass, Class IIb microcins, mediates Enterobacteriaceae dynamics in vivo(12,13).

Class IIb microcins are ribosomally synthesized bacteriocins between 5 kDa to 10 kDa in size with activity against closely related strains or species(13–17). Unlike all other microcins, they carry a serine-rich C-terminal motif for a posttranslational modification with a siderophore, here an enterobactin or an enterobactin derivative, before they are secreted into the extracellular space(18). Siderophores are iron-chelating molecules commonly employed by various bacteria to scavenge free iron to outgrow other bacteria, particularly in resource-scarce environments such as the gastrointestinal tract(15,16,19) and are often associated with increased pathogenicity or virulence(20–22). The iron chelating moiety of these siderophore antimicrobial peptides (sAMPs) is recognized by high-affinity receptors and functions as a ‘Trojan Horse’ key to susceptible bacteria as it triggers import into the periplasmic space, where the peptide inhibits the molecular target of susceptible bacteria(19,23–25). Because of these features, delivery of class IIb microcins by wildtype and engineered probiotics has been recently proposed as a strategy to combat drug-resistant enteric bacteria(12,13,16,26).

To date only five class IIb microcins have been described and only four have been characterized in terms of their antimicrobial activity. Specifically, the sAMPs MccE492 and MccG492 (uncharacterized) are solely present in Klebsiella pneumoniae (Kp), whereas MccH47 is specific for Escherichia coli (Ec)(15). Additionally, truncated versions of mciA (MccI47) and mcmM (MccM) are present in Kp RYC492, whereas they are intact in the Ec CA46 genome(15). Interestingly, while the genes encoding for microcins posttranslational modifications are highly conserved between Ec and Kp, suggesting a conserved pathway for microcin maturation, the toxin and corresponding immunity genes are significantly more variable (Figure S1). We hypothesized that class IIb microcin production extends beyond these specific compounds and organisms and identified a total of twelve novel class IIb microcins in seven additional Enterobacteriaceae species. In inhibition assays utilizing heterologous expression of these compounds, we demonstrate potent activity against a library of bacteria, including gram-negative ESKAPE and plant pathogens, .proving that class IIb microcins are more prevalent and more broad-spectrum in the natural microbial world than previously recognized.

Results

With the hypothesis that class IIb microcin production is a common trait among Enterobacteriaceae, we anticipated that the genes encoding for the antimicrobial and immunity would exhibit a high degree of dissimilarity to already known peptides as target specificity may result in accelerated adaptive coevolution(27). Therefore, in addition to the known microcin and immunity genes from MccE492, MccG492, MccH47, MccI47, and MccM, in our informatic approach we included the genes that are necessary for mature class IIb microcin biosynthesis, extending our search to longer sequences for more reliable Basic Local Alignment Search Tool (BLAST) results(28). Moreover, we hypothesized that the amino acid sequences of genes responsible for posttranslational modification and microcin export would be less prone to evolutionary changes, thereby maintaining the functional integrity of the gene cluster(15,29). We then assessed their proximity in the respective genome location, because microcin genes are typically flanked by genes essential for toxin maturation(15). Further, we assessed small open reading frames upstream and downstream of the maturation genes, allowing us to identify novel class IIb microcins without significant sequence similarity to the known antimicrobials, enabling the discovery of compounds with new molecular targets or modes of action (see Methods).

Our informatics-driven analysis identified twelve promising class IIb microcin candidates from seven gene clusters with high similarity to Ec CA46 and Kp RYC492 in seven species across the Enterobacteriaceae family (Figure 1A, Figure S2): (i) Brenneria goodwinii (Bg; 2; GenBank: CP014137), (ii) Gibbsiella quercinecans (Gq; 1; CP014136), (iii) Klebsiella oxytoca (Ko; 1; CP033844), (iv) Pantoea sp. (Ps; 1; CP034363), (v) Raoultella ornithinolytica (Ro; 4; CP008886), (vi) Salmonella enterica (Se; 2; CP030220), (vii) Serratia fonticola (Sf; 1; CP033055). Remarkably, although it has traditionally been a defining characteristic of class IIb microcins that all required genes are encoded within the chromosome(12), the gene cluster we discovered for Se is situated on a 159 kbp plasmid. Phylogenetic sequence analysis of both the antimicrobial and immunity peptide genes revealed the presence of eight different clades represented in both trees, respectively (Figure 1B,C). Regarding the well-established class IIb microcins MccH47, MccI47, MccM, MccG492, and MccE492, we identified novel members for each group, supported by sequence similarity, amino acid identity, the closest BLAST match, and domain predictions (Table 1, Figure S3). In light of this discovery, we propose a new nomenclature for class IIb microcins that includes the species initials in which they were identified (e.g., Ec, Kp), the closest relative already characterized class IIb microcin (G492, E492, H47, I47 or M), as well as the identifiers ‘A’ for antimicrobial or ‘I’ for immunity gene.

View this table:
Table 1: Blastp results and closest matches to the known class IIb microcins MccE492, MccG492, MccH47, MccI47, or MccM.

Red color indicates no significant match found.

Figure 1:
Figure 1: Novel class IIb microcins are found in numerous Enterobacteriaceae genomes.

(A) Sequence alignments of the newly identified microcin and immunity genes with the gene clusters of Ec CA46 and Kp RYC492 using Easyfig(30). Antimicrobial (A) and immunity (I) genes in the center are represented by darker and lighter shades, respectively. X=mchX, I=mchI, B=mchB, E=mceE, L=mceL, M=mceM. (B,C) Phylogenetic trees of antimicrobial and corresponding immunity genes using codon-aligned nucleotide sequences with General Time Reversible model with discrete gamma distribution (GTR+G) and the Hasegawa-Kishino-Yano model with discrete gamma distribution (HKY+G), respectively. (D) MUSCLE(31) alignment of the amino acid sequence of the signal peptide sequence as well as the C-terminus of the antimicrobial peptides.

Thus, the novel G492 relative found in Salmonella enterica will be called Se G492 with the antimicrobial peptide identified as Se G492A and the immunity peptide identified as Se G492I. It is worth highlighting that in the case of the G492 group, all its members have the immunity gene located downstream of the antimicrobial gene, whereas for the other clades, this arrangement is reversed. In addition to uncovering eight novel variants of the five previously characterized microcins, we have identified four additional microcins that seem to belong to three entirely new clades. We name these microcins microcin W (MccW), microcin X (MccX), and microcin Z (MccZ). The two members of the microcin X group, found in B. goodwinii (Bg X) and R. ornithinolytica (Ro X), only show significant similarity between one another, but not to any of the other antimicrobial or immunity peptides (Figure 1 B,C, Table 1, Table S1). Similarly, MccW from Gibbsiella quercinecans (Gq W) does not show any sequence similarity to either the known or novel antimicrobial or immunity peptides in terms of sequence similarity, amino acid identity, the respective BLAST hits (Table 1, Table S1), or phylogenetic localization (Figure 1 B,C). Lastly, MccZ from R. ornithinolytica (Ro Z) shows insignificant similarity with Ec MA (mcmA) for the antimicrobial, whereas the immunity peptide does not have any match among the known or the novel microcins (Table 1, Table S1). Crucially, the identification of MccX, and MccZ within the same gene clusters as representatives of the E492 (Bg E492), H47 (Ro H47), and I47 (Ro I47) groups strongly implies that they are functional components of a gene cluster.

To test the newly identified microcins for antimicrobial activity in our previously established Ec overexpression system(13,16), all antimicrobial and immunity peptides were codon optimized, synthesized, and cloned into an inducible high copy vector (see Methods). Through static plate inhibition assays involving live-producing cells(13,16,26), we successfully validated the robust antimicrobial activity of eleven out of the twelve newly discovered microcins (Figure 2A). These microcins exhibit a range of specificities, with some inhibiting targets narrowly (e.g., Ps G492AI), while others exert a broader effect against multiple bacteria (e.g., Se G492AI). Notably, our study also provides the first evidence of inhibitory activity by Kp G492, a microcin whose existence and function have only been proposed in the scientific literature based on genetic sequence(15).

Figure 2:
Figure 2: Novel sAMPs are effective inhibitors of Enterobacteriaceae and gram-negative ESKAPE pathogens.

(A) Heatmap summarizing the inhibitory potential of known and novel class IIb microcins against a library of Enterobacteriaceae, Pseudomonadales, and gram-positive bacteria, including multidrug-resistant isolates (red) as determined by static inhibition assays with live producing bacteria. *=activity determined through microcin purification and minimum inhibitory concentration assays(13,16). (B) Static inhibition assays comparing Kp E492, Kp G492, Ec H47, Ec I47, Ec M, and Se G492 activity from single colony production against multidrug-resistant A. baumannii (BAA 1790) and P. aeruginosa (PA14). Note that iron-limited conditions (DP) are required for antimicrobial activity, confirming action of class IIb microcins. L-ara=L-arabinose, DP=2,2-dipyridyl, scale bars: 1 cm.

To this date class IIb microcins have been only shown to be very selective and only active against different species within the Enterobacteriaceae family(13,15,16,26). While the activity for the novel sAMPs varies, we here report, for the first time, antimicrobial activity outside of the Enterobacteriaceae family. Specifically, we found that microcins Ps G492 and Se G492 have activity against gram-negative multidrug-resistant ESKAPE pathogens with both being capable of inhibiting Acinetobacter baumannii (BAA 1790), and with microcin Se G492 alone also showing activity against Pseudomonas aeruginosa (PA14) (Figure 2).

Discussion

With a comprehensive analysis of publicly available bacterial genomes, we unraveled twelve previously undiscovered class IIb microcins. Among these findings, we identified three novel microcin clades, specifically MccW, MccX, and MccZ. Through heterologous expression, we showed antimicrobial activity for all but one novel microcins and are the first to demonstrate activity for the known class IIb microcin Kp G492. Hence, this research demonstrates that class IIb microcins exhibit a higher prevalence in natural settings than previously anticipated. As a result, their impact on ecological community dynamics in natural environments, including the growth of Pseudomonadales species, is likely broader than previously thought. Antimicrobial and immunity genes were overexpressed and tested in our Ec-derived expression system, which might have led to degrees of incompatibility between the antimicrobial peptides and genes for microcin maturation. Consequently, the process of posttranslational modification with the siderophore (i.e., monoglycosylated enterobactin) might be less efficient, and the export mediated by the native signal peptide less targeted, potentially leading to reduced observed activity in the static plate assays. Furthermore, it is important to note that these assays exhibit lower sensitivity compared to purification approaches with minimum inhibitory concentration (MIC) assays. Thus, the activity spectrums are likely to encompass a wider range than what has been described in this study. However, historically the microcin literature proves that ideal approaches for purification and MIC testing can vary between the antimicrobials(13,15,16,24).

Remarkably, we were able to expand the origins of class IIb microcins from the enteric bacteria Ec and Kp to other members of the Enterobacteriaceae family, including well-known phytopathogens(32–34). Specifically, B. goodwinii and G. quercinecans are associated with Acute Oak Decline (AOD) and are frequently isolated together(35). Similarly, the two strains containing microcin genes were isolated within the same research project. Notably, these bacteria grow synergistically(36), while upregulating iron transporters during co-culture(37), hinting at class Iib microcin-related competition. We were able to show activity of the overexpressed microcins against human-derived enteric isolates, however, their antimicrobial spectrum might have evolved to target more frequently encountered strains from the genus Brenneria or Gibbsiella. Further, we demonstrated activity of several class IIb microcins against the tree pathogens G. quercinecans, G. greigii as well as Rahnella victoriana(36,38,39). Thus, treatment with potent microcins, purified or produced in live bacteria, could present a viable option to target bacteria-caused plant diseases.

In health care settings the burden by gram-negative ESKAPE pathogens and multidrug-resistant Enterobacteriaceae weighs heavily on modern medicine and novel antimicrobials are needed to develop new treatment options(40,41). Antimicrobial peptides and particularly microcins are promising candidates for selective eradication of enteric pathogens and have been demonstrated to potently reduce pathogen colonization in vivo, when produced by a live probiotic(12,13). Here we present the most comprehensive library of class IIb microcins created so far, that is suited for heterologous expression and in vivo application for the development of novel live biotherapeutic products against drug-resistant enteric bacteria and gram-negative ESKAPE pathogens.

In this study, we challenge the prevailing notion that class IIb microcin production is limited to Ec and Kp. Through comprehensive genomic analysis of publicly available bacterial genomes, coupled with heterologous overexpression, we unveiled a set undiscovered class IIb microcins across Enterobacteriaceae species. Our findings not only expand the known repertoire of class IIb microcins but also hold significant implications. We demonstrate that these newly identified class IIb microcins exert remarkable inhibitory effects on ESKAPE pathogen species not affected by any of the current ones. This discovery underscores their potential as agents against a broader spectrum of pathogens, including those affecting humans and plants, thus opening new avenues for antimicrobial research and applications.

Materials and Methods

Bioinformatic class IIb microcin identification

We developed a pipeline that by leveraging BLAST(28) enabled us to mine publicly available genome databases for novel, previously undescribed sAMPs. We included mchCDEF and mcmL for Ec as well as mceCDGHIJ for Kp for posttranslational modification and export, expecting more reliable hits for longer and functionally conserved proteins in close proximity to class IIb microcin and immunity genes. Thus, we first ran tblastn(28) against RefSeq(42), to screen for all genes related to biosynthesis pathways, known microcin genes, as well as immunity gene sequences exhibiting homology to the microcin gene clusters found in Ec CA46 and Kp RYC492. Homology to the microcin gene clusters were guided by BLAST parameters sseqid (Genome ID), pident (percentage of identical positions) along with sstart (start of alignment in Genome) and send (end of alignment position in Genome). Resulting hits were concatenated by genome ID and assessed for their proximity to one another in the genome. These gene clusters should, at best, contain all the known genes required for toxin maturation, including mchCDEF and mcmL(15,16). In addition to genomic hits to the known microcins, small ORFs of 50 to 150 amino acids in size close to the biosynthesis genes were annotated and their domains were predicted using SMART(43). The ORFs were meticulously examined and assessed against established class IIb microcin criteria: (i) a serine-rich C-terminus culminating in a final serine, (ii) the presence of fewer than one cysteine residue, (iii) a signal peptide within the initial 15 amino acids ending with GG or GA, and (iv) close proximity (≤200 bp) to an ORF featuring a predicted transmembrane domain, typically encoding an immunity peptide. The identified genes were included in the pipeline’s input to expand the scope of gene detection. We repeated this process iteratively through the pipeline until no additional genes were added to the output. Subsequently, blastp was used to assess microcin similarity shown in Table 1 and Table S1.

Phylogenetic analyses

For the native full length coding sequence of the microcin and immunity genes a codon-based sequence alignment was generated using the MUSCLE algorithm(31). Then, we determined the best fit substitution models for maximum likelihood phylogenetic analyses, resulting in the General Time Reversible model with discrete gamma distribution (GTR+G) and the Hasegawa-Kishino-Yano model with discrete gamma distribution (HKY+G), respectively. A bootstrap test with 1000 replicates for maximum likelihood and random seed was conducted for both trees. Alignment, model testing, and tree building was performed in MEGA11(44).

Plasmids and heterologous class IIb microcin expression

ORFs of identified microcin and immunity genes were codon optimized for frequent Ec codon usage without creating repetitive sequences and synthesized by Integrated DNA Technologies (Coralville, IA) with 18 bp of native 5’ upstream sequence and 20 bp of native 3’ downstream sequence, respectively. Using Gibson Assembly(45), the genes were cloned into our previously established Ec class IIb microcin expression system that results in mature class IIb microcins posttranslationally modified with a monoglycosylated enterobactin(13,16). Briefly, the antimicrobial and the immunity genes are co-expressed under the control of an arabinose-inducible pBad/araC promoter in a high copy plasmid with a pUC-derived origin of replication. All assemblies were verified using whole plasmid sequencing. DNA files for all used plasmids can be found at:

Static inhibition assays

Cultures of strains with confirmed plasmid assemblies were spread in LB agar plates containing 100 μg/ml ampicillin. Single colonies for each microcin were picked with a sterile pipet tip and inserted into solid LB agar containing 100 μg/ml ampicillin for plasmid retention, 0.2 mM 2,2-dipyridyl to create iron limited conditions during the growth phase, and 0.4% L-arabinose for induction of gene expression. Plates were incubated at 37°C for up to 72 h, before they were overlaid with the target bacterial isolates. For the overlay, the microcin-producing bacteria in the stabs were inactivated using chloroform vapors and ten minutes under ultraviolet light. Then, target bacteria were diluted 1:2000 from overnight culture in LB media containing 100 μg/ml ampicillin and 0.2 mM 2,2-dipyridyl. Ec and S. flexneri strains were diluted 1:200 to acquire dense bacterial lawns. Finally, 0.5 ml of molten agar was added to the liquid media and the resulting soft agar was spread on the plate with the inactivated bacteria and incubated for 16 h at 37°C.

Availability of Data and Materials

The genomes are accessible with the following GenBank numbers: Brenneria goodwinii (GenBank: CP014137), Gibbsiella quercinecans (CP014136), Klebsiella oxytoca (CP033844), Pantoea sp. (CP034363), Raoultella ornithinolytica (CP008886), Salmonella enterica (CP030220), Serratia fonticola (CP033055). All all information is included in the manuscript or supporting files. All plasmid sequences as well as annotation files to produce Figures 1A, S1, S2, and S3 are available as supplementary material.

Funding Disclosure

This work was supported partially supported by the CDMRP PRMP W81XWH2020013 to V.B., by the NIH NIA 1R01AG075283-01A1 to V.B., and by the Deutsche Forschungsgemeinschaft (DFG) project 457837076 to B.M.M.

Competing Interests

V.B. receives support from a sponsored research agreement from Vedanta Biosciences, Inc.

Author Contributions

B.M.M, S.K.B, and V.B conceptualized the study. S.K.B. performed bioinformatic data mining. B.M.M and V.B designed the experiments. B.M.M performed plasmid design, verification, and in vitro activity testing. B.M.M, S.K.B, and V.B wrote the manuscript.

Acknowledgements

The author would like to thank the administrative staff in the Microbiology and Physiological Systems Department at the University of Massachusetts Chan Medical School, especially Annette Bohigian, Amy Parker, Dhruti Desai, Marie Berardi, Richard Fish, and Tracey Rae, for their support.

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Expanding the toolbox: Novel class IIb microcins show activity against gram-negative ESKAPE and plant pathogens
Benedikt M. Mortzfeld, Shakti K. Bhattarai, Vanni Bucci
bioRxiv 2023.12.05.570296; doi: https://doi.org/10.1101/2023.12.05.570296
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