The early stage peptidoglycan biosynthesis Mur enzymes are antibacterial and antisporulation drug targets for recurrent Clostridioides difficile infection

Journal Pre-proof The early stage peptidoglycan biosynthesis Mur enzymes are antibacterial and antisporulation drug targets for recurrent Clostridioides difficile infection Madhab Sapkota, Ravi K.R. Marreddy, Xiaoqian Wu, Manish Kumar, Julian G. Hurdle PII:

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DOI:

https://doi.org/10.1016/j.anaerobe.2019.102129

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Received Date: 31 May 2019 Revised Date:

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Please cite this article as: Sapkota M, Marreddy RKR, Wu X, Kumar M, Hurdle JG, The early stage peptidoglycan biosynthesis Mur enzymes are antibacterial and antisporulation drug targets for recurrent Clostridioides difficile infection, Anaerobe (2019), doi: https://doi.org/10.1016/j.anaerobe.2019.102129. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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The Early Stage Peptidoglycan Biosynthesis Mur Enzymes Are Antibacterial

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and Antisporulation Drug Targets for Recurrent Clostridioides difficile

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Infection

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Madhab Sapkota1*, Ravi K. R. Marreddy2*, Xiaoqian Wu2, Manish Kumar1#, Julian G. Hurdle2

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University of Texas at Arlington, Department of Biology, Arlington, Texas, United States

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760191, Texas A & M University Health Science Center, Biosciences and Technology, Houston,

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Texas, United States 770302

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*Both authors equally contributed.

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#

Current address: Department of Biology, Texas State University, San Marcos, Texas, United

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States 78666

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Corresponding author: Julian G. Hurdle. E-mail address: [email protected]

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Abstract

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Sporulation during Clostridioides difficile infection (CDI) contributes to recurrent disease. Cell

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division and sporulation both require peptidoglycan biosynthesis. We show C. difficile growth

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and sporulation is attenuated by antisenses to murA and murC or the MurA inhibitor fosfomycin.

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Thus, targeting the early steps of peptidoglycan biosynthesis might reduce the onset of recurrent

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CDI.

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Keywords: Peptidoglycan, Mur ligases, antibiotic drug-targets heat-resistant spore cortex

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Antibiotic options to treat Clostridioides difficile infections (CDI) are fidaxomicin, vancomycin

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and metronidazole [1]. However, 20% or more of patients treated for CDI go on to experience

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recurrence of diarrhea following treatment [2, 3]. Following treatment, the failure of the normal

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gut microbiota to repopulate the large bowel is thought to allow C. difficile to recolonize the

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bowel and cause recurrent CDI (rCDI). In this regard, during infection, the formation and

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survival of spores that are intrinsically resistant to antibiotics is a major contributor to rCDI and

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transmission of the disease [4]. It is estimated that half or more of the cases of rCDI is due to

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endogenous spores, while reinfection with environmental spores account for other cases [5].

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Given the link between endogenous spores and rCDI, there is a need for therapeutics that

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block sporogenesis. Current efforts to find anti-sporulation drug targets focus on proteins that

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primarily affect sporogenesis, but are not required for vegetative growth [6, 7]. These ongoing

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efforts might lead to sporulation inhibitors that can serve as adjuvants to standard of care

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antibiotics. However, there is a continuing need for new anti-C. difficile antibiotics as resistance 2

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has emerged to all currently used first-line antibiotics [8, 9]. We propose that enzymes involved

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in cytoplasmic steps of peptidoglycan biosynthesis could be suitable antibacterial and

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antisporulation drug targets, because they play essential roles in vegetative cells and spores [9-

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12]. Peptidoglycan is critical for maintaining cell shape and rigidity to prevent osmotic lysis,

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while in sporogenesis, as seen in Bacillus spp., it enables mother cell engulfment of the forespore

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and eventual formation of the peptidoglycan spore cortex that confers the heat resistance of

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spores [10].

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Peptidoglycan biosynthesis is a multi-step process involving: (1) formation of N-

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acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) disaccharide pentapeptide

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peptidoglycan precursors; (2) transport across the cell envelope and (3) assembly of the

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precursors into the growing peptidoglycan layer. Among the enzymes required for synthesis of

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the disaccharide pentapeptide are the Mur enzymes. Furthermore, in B. subtilis, depletion of the

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Mur enzyme UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) decreases sporulation

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and causes cells to be more sensitive to cell wall synthesis inhibitors [10]. We therefore

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investigated Mur enzymes, UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) and

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UDP-N-acetylmuramate-L-alanine ligase (MurC), which catalyze the first and third steps of

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peptidoglycan precursor biosynthesis [13]. Many Gram-positive bacteria harbor two copies of

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murA [14], but C. difficile expresses a single murA that is predicted to be located in an operon

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(Figure S1) with sporulation genes SpoIIC (Stage II sporulation protein D) and SpoIIID (Stage

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III sporulation protein D). The genetic arrangement of sporulation and cell wall biosynthesis

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genes in operons is also evident in B. subtilis [10] and in C. perfringens ATCC 13124 (accession

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no. NC_008261). Because C. difficile murC is monocistronic we selected it for antisense

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analysis, to confirm that anti-sporulation outcomes from murA depletion was not due to polar

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effects on downstream sporulation genes.

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Strains and plasmids used in this study are indicated in Table 1; primers and antisense

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sequences are in Table S1. Antisense RNAs (asRNAs) to murA, murC, spo0A were expressed

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under the anhydrotetracycline (ATc) inducible pTet promoter in plasmid pMSPT, which carries a

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paired terminus to stabilize the antisense RNA [15]. The asRNAs were 100 bp in length; for

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murA and spo0A targeted the upstream region of the gene, including the ribosome binding site,

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whereas that to murC targeted the middle region of the gene (Table S1). The antisense fragments

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to murA, murC and spo0A were cloned into XhoI and SphI sites of pMSPT. Constructs were

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conjugated into C. difficile CD630 as described [15]. Antisenses were induced with ATc.

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Susceptibility to fosfomycin and vancomycin were determined by microdilution in 96-well plate,

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using two-fold antibiotic dilutions and inocula of 105 CFU/ml of C. difficile. Antibiotic minimum

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inhibitory concentrations (MICs) were defined as the lowest concentration of compound

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inhibiting visible growth. Automated growth kinetics were performed in 96-well using

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logarithmic cultures in a Versa max microplate reader, which recorded OD600nm every 30 min

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for 24 h, with shaking before each read, in a Coy anaerobic chamber.

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Induction of asRNA to murA mRNA inhibited growth of C. difficile CD630 on agar in a

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dose-dependent manner at ATc concentrations of ≥0.004 µg/mL and above (Figure 1A);

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similarly, expression of asRNA to murC mRNA inhibited grown on agar with ATc at ≥0.008

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µg/mL (Figure 1A). In broth, strong dose-dependent growth inhibition was also seen for asRNAs

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to murA and murC (Figure 1B-C). The test concentrations of ATc did not inhibit growth of the

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control strain carrying the empty vector pMSPT (Figure 1A and S2). To analyze the effect of

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antisense fragments on RNA levels, RT-qPCR was performed on four biological replicates as 4

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described previously [15] and values analyzed by t-test in GraphPad prism 8. ATc concentrations

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were chosen to represent ½× and 4× the inducer concentration that results in growth inhibition,

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to assess dose response. As shown in Figure 1D, induction of asRNA to murA at 0.004 µg/ml of

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ATc reduced cDNA formation by 5.18 ± 2.6 (p = 0.0087) (Figure 1D), while a 8-fold increase in

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ATc to 0.032 µg/ml caused a 13.5 ± 8.1- fold reduction in cDNA (p = 0.0077). Transcript levels

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were unaffected for upstream (CD630_01220, encoding a hypothetical protein) and downstream

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(spoIIC, stage II sporulation protein D and CD630_01250, encoding a hypothetical protein)

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genes in the operon (Figure S3) and murC (Figure S3). This is consistent with inhibition of

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translation by asRNAs designed to resemble naturally occurring antisenses, as previously

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described [15]. Against murC, cDNA formation was reduced by 3.51 ± 1.7 fold (p = 0.0020)

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with ATc at growth inhibitory concentration of 0.008 µg/mL (Figure 1E); further reduction in

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cDNA formation (6.28 ± 3.6- fold; p = 0.0025) occurred at ATc 0.064 µg/mL. Expression of the

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antisense to murC did not affect murA transcript levels (Figure S4). Consistent with the effect of

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cell wall synthesis inhibition [16], antisenses to murA and murC caused cell elongation (Figure

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S5). As a further control, we tested spo0A, an early stage sporulation gene, which does not

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mediate vegetative cell growth; induction of asRNA to spo0A did not affect growth on agar

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(Figure 1A). Taken together these observations confirms the essentiality of MurA and MurC for

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vegetative growth of C. difficile.

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To study whether murA and murC were also essential for C. difficile spore formation, we

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analyzed sporulation in presence and absence of asRNA to respective genes. Sporulation was

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analyzed as described previously [15], using four biological replicates to determine the number

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of spores produced by cells at day 5. There was a significant reduction in sporulation by cells

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expressing asRNA to murA upon induction with 0.004 and 0.032 µg/mL of ATc; these 5

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concentrations represent ½× and 4× the inducer concentration that results in growth inhibition.

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Spore formation was reduced as the total population contained 8.8-18.6% of spores (Figure 2A),

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in contrast to the empty vector control in which spore formation was not inhibited by the

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respective ATc concentrations. Induction of asRNA to murC (0.008 and 0.064 µg/mL of ATc or

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½× and 4× the inducer concentration causing growth inhibition) also reduced spore as there were

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8.1-25.1% spores in the total population (Figure 2B). The percent of spores in the total viable

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population for asRNA to murA and murC were comparable to cells expressing asRNA to spo0A,

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which encode a key transcriptional regulator of the initial stages of sporulation (Figure 2C).

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To confirm findings from the above genetic studies, we tested the antibiotic fosfomycin,

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which inhibits MurA by covalently binding to a conserved cysteine in its active site. Fosfomycin

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at ¼× and 1× its MIC (8 µg/mL) reduced spore formation, as there were 11.3-14.7% of spores in

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the population (Figure 2D). Inhibition of sporulation by fosfomycin was comparable to the

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positive control acridine orange (AO). In contrast, vancomycin, which inhibits the final stage of

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cell wall assembly at the cell envelope, did not inhibit sporulation in cells exposed to ¼× and 1×

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its MIC (0.25 and 1.0 µg/mL). Sub-inhibitory fosfomycin (¼× MIC) did not affect toxin

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production (Figure S6). These findings further support the essentiality of MurA for sporulation

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and vegetative growth of C. difficile.

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Although traditional antibiotics metronidazole and vancomycin inhibit C. difficile

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growth, the growth inhibited cells are still capable of undergoing sporulation, which contrasts

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with the newer drug fidaxomicin that inhibits sporulation and toxin production by inhibiting gene

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transcription [17, 18]. Because alternative antibiotics will be needed to cover emerging

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antibiotic-resistant C. difficile, we reason that discovery efforts should focus on antibacterial

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drug targets that affect C. difficile sporulation and/or toxin production. Recently, we reported that 6

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sporulation is decreased upon inhibition of the enoyl ACP-reductase FabK enzyme, which

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catalyzes the final step of bacterial fatty acid biosynthesis [15]. We now show that MurA and

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MurC enzymes could also be potential drug targets for the discovery of anti-sporulation

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antibacterial agents. The enzymatic mechanisms of Mur ligases are well known, allowing for

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high throughput assays to identify enzyme inhibitors [19]. A potential challenge will be

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identifying compounds that selectively inhibit C. difficile, avoiding inhibition of gut microbiota

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that also carry these enzymes. However, narrow-spectrum inhibitors of C. difficile Mur ligases

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may be developed with an increase in current understanding of substrate transporters in C.

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difficile that are absent in key gut flora [20, 21]. This may allow engineering of antibiotics that

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use the designated transporters to reach their cytoplasmic targets. Additionally, Mur ligase

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inhibitors may be designed with physicochemical properties favoring their uptake into Gram-

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positive bacteria [22]. We anticipate that such inhibitors would be valuable in reducing the risk

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of rCDI.

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This study was in part supported by National Institute of Allergy and Infectious Diseases

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of the National Institutes of Health grant R21AI126755. MS acknowledges support from the

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University of Texas at Arlington.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at

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References

146

[1]

147

Clinical practice guidelines for Clostridium difficile infection in adults and children: 2017

148

Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare

149

Epidemiology of America (SHEA). Clin. Infect. Dis. 66 (2018) 987-94.

150

[2]

151

cure and increased recurrence rates for Clostridium difficile infection caused by the epidemic C.

152

difficile BI strain. Clin. Infect. Dis. 55 (2012) 351-7.

153

[3]

154

Burden of Clostridium difficile infection in the United States. N. Engl. J. Med. 372 (2015) 825-

155

34.

156

[4]

157

Clostridium difficile spo0A gene is a persistence and transmission factor. Infect. Immun. 80

158

(2012) 2704-11.

159

[5]

160

difficile recurrence. Clin. Microbiol. Infect. 24 (2018) 476-82.

161

[6]

162

the classic model. FEMS Microbiol. Lett. 358 (2014) 110-8.

163

[7]

164

SinR' regulates toxin, sporulation and motility through protein-protein interaction with SinR.

165

Anaerobe (2019).

166

[8]

167

national survey of the molecular epidemiology of Clostridium difficile in Israel: the

L.C. McDonald, D.N. Gerding, S. Johnson, J.S. Bakken, K.C. Carroll, S.E. Coffin, et al.

L.A. Petrella, S.P. Sambol, A. Cheknis, K. Nagaro, Y. Kean, P.S. Sears, et al. Decreased

F.C. Lessa, Y. Mu, W.M. Bamberg, Z.G. Beldavs, G.K. Dumyati, J.R. Dunn, et al.

L.J. Deakin, S. Clare, R.P. Fagan, L.F. Dawson, D.J. Pickard, M.R. West, et al. The

C.H. Chilton, D.S. Pickering, J. Freeman. Microbiologic factors affecting Clostridium

A.N. Edwards, S.M. McBride. Initiation of sporulation in Clostridium difficile: a twist on

Y. Ciftci, B.P. Girinathan, B.A. Dhungel, M.K. Hasan, R. Govind. Clostridioides difficile

A. Adler, T. Miller-Roll, R. Bradenstein, C. Block, B. Mendelson, M. Parizade, et al. A

8

168

dissemination of the ribotype 027 strain with reduced susceptibility to vancomycin and

169

metronidazole. Diagn. Microbiol. Infect. Dis. 83 (2015) 21-4.

170

[9]

171

Characterization of a clinical Clostridioides difficile isolate with markedly reduced fidaxomicin

172

susceptibility and a V1143D mutation in rpoB. J. Antimicrob. Chemother. 74 (2019) 6-10.

173

[10]

174

cluster is important for growth and sporulation. J. Bacteriol. 188 (2006) 1721-32.

175

[11]

176

Vannieuwenhze, et al. Peptidoglycan transformations during Bacillus subtilis sporulation. Mol.

177

Microbiol. 88 (2013) 673-86.

178

[12]

179

formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the

180

control of sigma K. Mol. Microbiol. 65 (2007) 1582-94.

181

[13]

182

ligases. J. Mol. Biol. 362 (2006) 640-55.

183

[14]

184

forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria. J.

185

Bacteriol. 182 (2000) 4146-52.

186

[15]

187

acid synthesis protein enoyl-ACP reductase II (FabK) is a target for narrow-spectrum

188

antibacterials for Clostridium difficile infection. ACS Infect. Dis. 5 (2019) 208-17.

J. Schwanbeck, T. Riedel, F. Laukien, I. Schober, I. Oehmig, O. Zimmermann, et al.

G. Real, A.O. Henriques. Localization of the Bacillus subtilis murB gene within the dcw

E.I. Tocheva, J. Lopez-Garrido, H.V. Hughes, J. Fredlund, E. Kuru, M.S.

P. Vasudevan, A. Weaver, E.D. Reichert, S.D. Linnstaedt, D.L. Popham. Spore cortex

C.A. Smith. Structure, function and dynamics in the mur family of bacterial cell wall

W. Du, J.R. Brown, D.R. Sylvester, J. Huang, A.F. Chalker, C.Y. So, et al. Two active

R.K.R. Marreddy, X. Wu, M. Sapkota, A.M. Prior, J.A. Jones, D. Sun, et al. The fatty

9

189

[16]

P. Nonejuie, M. Burkart, K. Pogliano, J. Pogliano. Bacterial cytological profiling rapidly

190

identifies the cellular pathways targeted by antibacterial molecules. Proc. Natl. Acad. Sci. U S A

191

110 (2013) 16169-74.

192

[17]

193

Fidaxomicin inhibits spore production in Clostridium difficile. Clin. Infect. Dis. 55 Suppl 2

194

(2012) S162-9.

195

[18]

196

and related phytochemicals as nature-inspired treatments for Clostridium difficile infection. J.

197

Appl. Microbiol. 116 (2014) 23-31.

198

[19]

199

antibacterial target. Mol. Microbiol. 94 (2014) 242-53.

200

[20]

201

Garcia-Angulo. Extensive identification of bacterial riboflavin transporters and their distribution

202

across bacterial species. PLoS One 10 (2015) e0126124.

203

[21]

204

Convergent loss of ABC transporter genes from Clostridioides difficile genomes is associated

205

with impaired tyrosine uptake and p-Cresol production. Front. Microbiol. 9 (2018) 901.

206

[22]

207

109.

F. Babakhani, L. Bouillaut, A. Gomez, P. Sears, L. Nguyen, A.L. Sonenshein.

X. Wu, M.Z. Alam, L. Feng, L.S. Tsutsumi, D. Sun, J.G. Hurdle. Prospects for flavonoid

I. Kouidmi, R.C. Levesque, C. Paradis-Bleau. The biology of Mur ligases as an

A. Gutierrez-Preciado, A.G. Torres, E. Merino, H.R. Bonomi, F.A. Goldbaum, V.A.

M. Steglich, J.D. Hofmann, J. Helmecke, J. Sikorski, C. Sproer, T. Riedel, et al.

L.L. Silver. Challenges of antibacterial discovery. Clin. Microbiol. Rev. 24 (2011) 71-

208

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Tables

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Table 1. Strains and plasmids used in this study. Strain

Plasmid

Description

Source

C. difficile CD630

pMSPT

pRPF185 derivative in which the gusA gene was replaced with paired termini sequence

[15]

C. difficile CD630

pMSPT-murAi

pMSPT containing antisense murA RNA

This work

C. difficile CD630

pMSPT-murCi

pMSPT containing antisense murC RNA

This work

C. difficile CD630

pMSPT-spo0Ai

pMSPT containing antisense spo0A RNA

[15]

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Figures and legends

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Figure 1

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Impact of antisenses to murA and murC on C. difficile growth and gene expression. (A) C.

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difficile strain carrying empty plasmid or plasmids encoding antisense RNA were analyzed on

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BHI agar. Three micro liters of overnight cultures were spotted on BHI agar plates containing

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required concentrations of anhydrotetracycline (ATc); representative results from >3

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independent experiments are shown. (B & C) Growth kinetics (n=4 biological replicates) for the

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strains were analyzed by microdilution in 96-well plate. Growth was analyzed for C. difficile

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CD630 strain harboring asRNA to murA (B) and asRNA to murC (C). The growth was analyzed

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at different concentrations of ATc (µg/ml) i.e. at 0 (black), 0.004 (green), 0.008 (orange), 0.032

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(red) and 0.064 (blue). (D & E) mRNA levels for murA (D) and murC (E) genes were analyzed

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by RT-qPCR on 4 biological replicates. The fold-change was calculated for difference in mRNA

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levels between strains containing empty vector and those with antisense constructs. Data in D

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and E were analyzed by t-test in GraphPad prism 8 and significance at p<0.01 is shown by

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asterisks.

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Figure 2

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Inhibition of murA and murC gene activity affects C. difficile sporulation. Spore production

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was analyzed at day 5, following induction by anhydrotetracycline (ATc) of antisenses to murA

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(A), murC (B) and spo0A (C). (D) Comparison of inhibition of sporulation by fosfomycin (FOS

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[MIC = 8 µg/mL]) and vancomycin (VAN [MIC = 0.25 µg/mL]). Acridine orange (AO) served

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as a positive control. Data from four biological replicates were analyzed by t-test in GraphPad

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prism 8 and significance at p<0.01 and p<0.001 are shown by asterisks.

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Highlights Early stage cell wall synthesis is essential for vegetative cells and sporogenesis. MurA and MurC catalyzes the first and third steps in cell wall synthesis. Growth and sporogenesis were affected by genetic knockdown of murA and murC genes. Sporogenesis was inhibited by the antibiotic fosfomycin, targeting MurA. Recurrent C. difficile infection (rCDI) is linked survival of endogenous spores. Early stage cell wall synthesis enzymes are potential drug targets to prevent rCDI.

Declaration of interest None to declare.