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Noda Institute for Scientific Research GRANT2014 Young Investigator Research GrantOutline of Research Result

Identification of the Staphylococcus aureus exotoxintransporterChikara KaitoLaboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo

Research aimsDrug pumps contribute to the multi-drug resistance ofMRSA, Pseudomonas aeruginosa, and Acinetobacter baumannii, and thus understanding their functions is importantto establish clinical treatment strategies against MRSA andother antibiotic-resistant pathogenic bacteria.
S. aureus AbcA comprises six transmembrane domainsand an ATP hydrolyzing domain in a single polypeptide(575 aa), which is a type III ABC transporter found in theexport system of many bacteria. Overexpression of abcAleads to resistance against β-lactams, such as methicillinand cefotaxime; the phosphoglycolipid, moenomycin; thelipopeptide antibiotic, daptomycin; and dyes, such as rhodamine and ethidium bromide. These findings suggest thatAbcA is a multidrug efflux system in S. aureus. AbcA expression is regulated by NorG, a transcription factor involved in antibiotic resistance, and other virulence regulators, including AgrBDCA, Rot, SarA, SarZ, and MgrA. Additionally, AbcA expression is induced by antibiotic-treatment and in starvation conditions or the stationary phase,wherein several S. aureus virulence factors are produced.
These findings suggest that AbcA plays a role in S. aureusvirulence in addition to drug efflux. However, the endogenous molecule exported by AbcA and the exact functions ofAbcA in S. aureus virulence have not been elucidated.
S. aureus produces phenol-soluble modulins (PSM) having high lytic activity against neutrophils and erythrocytes.
Additionally, deletion of genes encoding PSM decreases S.
aureus virulence in a mouse infection model. PSM is considered to be a determinant of the high virulence of community-acquired MRSA (CA-MRSA). Moreover, PSM isinvolved in in S. aureus colony spreading.1), 2) Therefore,elucidating the secretory mechanism of PSM is importantfor understanding the molecular mechanisms underlying S.
aureus infection.
PSM peptides do not have signal peptides recognized bySec machinery, but its characteristics are similar to those ofbacteriocins, which are antimicrobial peptides produced byvarious bacteria. Some S. aureus strains produce bacteriocins, namely Aureocin A70, with an isoelectric point of9.9–10.0. AurT(571 aa)-secreting Aureocin A70 is a typeIII ABC transporter comprising six transmembrane domains and an ATP hydrolyzing domain. We hypothesizedthat the transporter secreting PSM has a structure similar tothat of AurT, and found that AbcA shared 58% amino acididentity with AurT.
Methods and ResultsAbcA is required for PSM secretionWe searched for a protein similar to AurT from the S. aureus N315 genome database using protein BLAST andidentified AbcA (SA0599), which had the highest homology with AurT among S. aureus gene products. In addition,we found 17 S. aureus gene products having similarity toAbcA. We constructed knockout mutants of the S. aureusNewman strain for these genes and measured the amount ofPSM in culture supernatants. AbcA shared high identitywith AurT in the transmembrane domain, Walker A andWalker B motifs that are predicted to bind ATP, the signature C motif, and the switch region, which are all conservedamong bacterial ABC exporters. The amount of PSM in theculture supernatant of the abcA knockout mutant was lessthan that of the parent strain. This amount was restored byintroducing the intact abcA gene. The growth of the abcAdisrupted mutant was indistinguishable from that of the parent strain. We hypothesized that PSM would accumulate inthe cytosolic fraction of the abcA knockout mutant if PSMsecretion was blocked. In agreement, the amount of PSMwas higher in the intracellular fraction of the abcA knockout mutant than in that of the parent strain. This amount decreased with the introduction of the intact abcA gene in theabcA knockout mutant. These findings suggest that AbcA isrequired for PSM secretion.
The ATP binding motif of AbcA is required for PSM secretionBecause the ABC transporter utilizes energy released byATP hydrolysis, amino acid substitutions in the Walker A orWalker B motifs, which function in ATP hydrolysis, resultin the loss of the transporting activity of the ABC transporter. We constructed mutants encoding AbcA proteinsharboring an amino acid substitution in the Walker A orWalker B motifs. We examined their complementation activity in the abcA knockout mutant. Introduction of thewild-type abcA gene restored the amount of PSM in theculture supernatant of the abcA knockout mutant to the parent level. In contrast, mutant abcA genes encoding K374Ror D497R mutant proteins decreased the restoration activi-

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strain transformed with an empty vector. These findingssuggest that heterologously expressed AbcA protects L. lactis from extracellularly administered PSM.
ConclusionFig. 1. AbcA has dual functions

ties compared with the wild-type abcA gene. The expression levels of mutant AbcA proteins were comparable withthat of wild-type AbcA protein. These findings suggest thatthe ATP binding motif of AbcA is required for PSM secretion.
AbcA is required for S. aureus virulenceBased on the requirement of AbcA for PSM secretion,we investigated whether AbcA has a critical role in S. aureus virulence by using a murine systemic infection model.
The survival rate of mice injected with the abcA knockoutmutant was higher than that of mice injected with the parent strain. This finding indicates that AbcA has a criticalrole in S. aureus infection in mammals.
Heterologous expression of AbcA in L. lactis increasesresistance against PSMIf AbcA directly secretes or extrudes PSM, it is assumedthat heterologous expression of AbcA functions in protecting the bacteria from PSM. We thus examined whether heterologous expression of AbcA makes L. lactis resistant toPSM. When PSM was added to the culture, L. lactis strainexpressing AbcA had a higher survival rate than L. lactis

Our findings revealed that a multidrug resistance transporter, AbcA, is required for the secretion of the S. aureuscytolytic toxin PSM. Heterologous expression of AbcA inL. lactis protected the cells against extracellular PSM.
Therefore, our results suggest that AbcA secretes self-synthesized PSM as well as extrudes extracellularly administered PSM. Furthermore, an in vivo infection experimentdemonstrated that AbcA plays a role in S. aureus virulence.
These findings provide the first evidence that a multidrugtransporter has dual functions: to secrete toxins as well asto protect cells from foreign substances. Thus it possessesoffensive and defensive functions, both of which are important for the bacterial infection3) (Fig. 1).
The present study revealed that a drug resistance transporter secretes S. aureus cytolytic toxins, thus the link between drug resistance and virulence expression. Transporters are typically located on the cell surface, which is easilyaccessed by chemicals. Thus, inhibition of S. aureus AbcAmay lead to increased sensitivity to antibiotics and decreased virulence, making AbcA a promising target fornovel antibiotics against S. aureus.
References1)2)3)

Kaito C, et al. (2011) Transcription and translation products of thecytolysin gene psm-mec on the mobile genetic element SCCmecregulate Staphylococcus aureus virulence. PLoS Pathog. 7:e1001267.
Omae Y, Sekimizu K, Kaito C. (2012) Inhibition of colony-spreading activity of Staphylococcus aureus by secretion of δ-hemolysin.
J Biol Chem. 287: 15570-9.
Yoshikai H, Kizaki H, Saito Y, Omae Y, Sekimizu K, Kaito C.
(2016) Multidrug-resistance transporter AbcA secretes Staphylococcus aureus cytolytic toxins. J Infect Dis. 213: 295–304.