Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci

A truncated derivative of the phage endolysin LysK containingonly the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase)domain exhibited lytic activity against live clinical staphylococcalisolates, including methicillin-resistant Staphylococcus aureus.This is the first known report of a truncated phage lysin whichretains high lytic activity against live staphylococcal cells.

INTRODUCTION

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INTRODUCTION

Staphylococcus aureus is associated with a variety of clinicalmanifestations, ranging from skin infections to more acute conditionssuch as necrotizing pneumonia and septicemia (20). Since methicillin-resistantS. aureus (MRSA) is now the most commonly reported antibiotic-resistantbacterium in clinical settings (5), the development of alternativeantimicrobials is warranted.

Bacteriophage-encoded endolysins are a group of enzymes thatact by digesting the peptidoglycan of bacterial cell walls.The potential of these molecules for controlling bacterial infectionsand preventing the pathogenic colonization of mucosal membraneshas been demonstrated previously (2, 6, 10, 11, 14, 15, 21,23, 29). In general, phage endolysins have a modular organizationwith an N-terminal catalytic domain and a C-terminal cell-bindingdomain (9, 12, 18). To our knowledge, only a few phage endolysins,such as LysK, phi11, MV-L, and LysH5, have been reported tolyse live staphylococcal cultures (4, 25-27). LysK has a modularstructure similar to the structure of these endolysins, withtwo catalytic domains, a CHAP (cysteine- and histidine-dependentamidohydrolase/peptidase) domain and a central amidase-2 domain(N-acetylmuramoyl-L-alanine amidase), as well as a C-terminalSH3b cell-binding domain (4, 22, 25-27).

In this study, we examined the involvement of each of the threedomains of LysK during exolysis by performing a deletion analysisand identified truncated LysK proteins containing only the CHAPdomain, which still showed lytic activity against live clinicalstaphylococcal isolates, including MRSA. In previous studiesother workers have obtained similar results upon deletion ofthe cell wall-binding domains (1, 8, 16, 17, 19). Constructionof a single-domain protein for therapeutic purposes is desirablesince, as well as facilitating protein production, this maydecrease the likelihood of a significant immunogenic response.Unlike antibiotics, intact endolysins are large proteins whichare capable of stimulating a humoral immune response, especiallywhen they are used intravenously (7, 24, 30).

Generation of deletion derivatives of LysK.

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Generation of deletion...

LysK protein deletion mutants were constructed based on thedomain organization of this protein. The primer pairs used fordomain deletion analysis were FLysK plus Rami (CHAP and amidase-2),Fami plus RSH3b (amidase-2 and SH3b), Fami plus Rami (amidase-2),FSH3b plus RSH3b (SH3b), and FLysK plus R313 to R156 (CHAP)(Table 1). Amplified products were cloned into the pQE60 (Qiagen)expression system and transformed into Escherichia coli XL1-Bluefor expression. Cells were induced as previously described (3).However, induction was performed at 26°C for 14 h to avoidinclusion bodies.

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TABLE 1. Oligonucleotides used for construction of plasmids

CHAP domain exhibits lytic activity against heat-killed staphylococcal cells.

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CHAP domain exhibits lytic...

Preparation of protein samples, sodium dodecyl sulfate-polyacrylamidegel electrophoresis, and zymographic analysis were performedas previously described (13, 26). Zones of lysis on the zymogramgel were used as an indication of putative catalytic activity.The results suggested that in the absence of other domains,the LysK CHAP domain retains lytic activity, while the amidase-2and SH3b domains appear to have no significant lytic activitywhen they are expressed alone or in combination (Fig. 1).

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FIG. 1. Schematic diagram of LysK and its truncated derivatives. LysK (495 residues) contains a CHAP domain (residues 35 to 160), an amidase-2 domain (residues 197 to 346), and an SH3b domain (residues 412 to 481). Activity was determined using zymogram analysis with heat-killed MRSA strain DPC5645. The dotted line indicates the end of the CHAP domain. The numbers are the positions of the first and last amino acids. +, zone of clearing; –, no zone of clearing; +*, very faint zone of clearing.

To determine the smallest fully functional catalytic regionof LysK, a sequential deletion analysis of the CHAP domain wasperformed (Fig. 1, plasmids C203 to C156). Zymogram analysisindicated that while there was some residual activity with ashorter variant with only 159 amino acids (C159), full endopeptidaseactivity required amino acids 1 to 162 (C162), suggesting thatthe N-terminal CHAP domain is sufficient for lysis of S. aureus,including MRSA. In addition, the truncated derivatives showeda spectrum of inhibition similar to that of LysK, lysing allS. aureus strains tested, including MRSA, heterogeneous vancomycin-intermediateS. aureus, and other antibiotic-resistant variants (data notshown), but not lysing members of genera other than Staphylococcusamong the strains tested.

CHAP domain exhibits lytic activity against live cells.

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The native LysK protein and two derivatives of interest, C203(CHAP and six residues of amidase-2) and C165 (CHAP) (Fig. 1),were selected to determine the lytic activity against live staphylococcalcells. LysK and C165 were purified by ion-exchange and sizeexclusion chromatography, while C203 was purified by nickelaffinity chromatography (unpublished data). The specific activityof each protein was estimated as previously described (2, 23),with some modifications. Briefly, MRSA strain DPC5645 (MooreparkFood Research Centre Culture Collection) was grown to an opticaldensity at 590 nm (OD₅₉₀) of 0.3, centrifuged, and then resuspendedto a final OD₅₉₀ of 0.8 in 50 mM sodium acetate buffer (pH 6.5).Serial dilutions of 100 µl purified lysin were mixed with100 µl of the bacterial suspension and incubated at 37°C.The amount of lysin that reduced the OD₅₉₀ by 50% in 15 minwas defined as 1 U of activity. The protein concentration wasmeasured using a Bradford protein assay kit (Bio-Rad). Basedon the data obtained, the specific activities of LysK, C165,and C203 were calculated to be 34 U nmol^–1 (621 U mg^–1),68 U nmol^–1 (3,690 U mg^–1), and 2.4 U nmol^–1(100 U mg^–1), respectively. Increases and decreases inactivity were determined as previously described (1). Comparedto the activity of LysK, the activity of C165 was approximatelytwofold higher, demonstrating that a LysK derivative containingonly the CHAP domain is more active against live staphylococcalcells than the native enzyme. By contrast, the activity of C203was approximately 14-fold lower, which may have been due toa change in protein folding. It should also be emphasized thatwhile C165 did not contain a His tag, the native protein andC203 did contain such a tag, which potentially could have alteredits specific activity. Nonetheless, the results show that C165had high activity in this case. To compare the catalytic activities,DPC5645 cells were treated with the same quantity (0.5 nmol)of LysK, C203, and C165 (Fig. 2).

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FIG. 2. Comparison of lytic activities of purified LysK, C203, and C165 determined by using live DPC5645 cells in vitro. The amount of purified C203 (x), LysK ( ${blacksquare}$ ), or C165 ( ${blacktriangleup}$ ) used was 0.5 nmol. Control experiments (•) were performed under the same conditions with no enzyme added. The error bars indicate standard errors of the means.

LysK (26), phi11 (4), MV-L (27), and LysH5 (25) are the onlystaphylococcal endolysins that have been reported to kill untreatedstaphylococcal cells. However, deletion of additional domainsof phi11 changed it to a barely active lysin (4, 28), and whilethe LysK derivatives lyse cells of all members of the genusStaphylococcus, MV-L and LysH5 have only been shown to lyseS. aureus, as well as Staphylococcus simulans and Staphylococcusepidermidis, respectively. We determined that the CHAP domainalone (C162, 33%) can exhibit activity against live staphylococci.This phenomenon was also observed with other staphylococcalphage endolysins with a similar structure, such as PlyTW (24)and Ply187 (23), but lysis was observed with only heat-killedstaphylococcal cells. The LysK CHAP domain provides a valuablefunctional unit for domain-swapping studies. It would be interestingto investigate if a chimeric protein with the LysK CHAP domainand a different substrate-binding domain would have an alteredspectrum of inhibition, since we demonstrated that the CHAPdomain alone has the same spectrum of inhibition in all of thestrains tested. Environments such as hospitals and nursing homesin which there are high numbers of MRSA infections could benefitconsiderably from exploitation of the CHAP domain of LysK.

ACKNOWLEDGMENTS

We thank Susan Mills and David Donovan for their scientificadvice and Kieran Kilcawley for his technical assistance.

This research was funded by Teagasc and Science Foundation,Ireland. M. Horgan was the recipient of a Teagasc Walsh Fellowship.

FOOTNOTES

* Corresponding author. Mailing address: Biotechnology Centre, Teagasc, Moorepark Food Research Centre, Fermoy, County Cork, Ireland. Phone: 353 (0)25 42229. Fax: 353 (0)25 42340. E-mail: paul.ross{at}teagasc.ie

Published ahead of print on 1 December 2008.

REFERENCES

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