Introduction:

Upon bacterial infection, the bacteria should sense and respond appropriately to environmental conditions in hosts; nutrient concentration, pH and to the defense system deployed by the host such as the antimicrobial peptides, complement system, opsonins or other components, which interact with the cell wall and/or bacterial membrane. To respond to these conditions, the membrane associated-proteins including nutrient receptors and two-component sensor-kinase and regulator systems play important roles. Virulence of pathogenic bacteria is also largely determined by membrane-associated proteins and secreted toxins. Therefore, the integrity of the membrane and/or cell wall of bacteria should be consequently required for the efficient function of these response and virulence determinants.

In this study, based on the standpoint that the virulence of S. aureus could be impaired by the dysfunction of some membrane-associated proteins, I searched for the virulence genes.

Identification of an S. aureus gene responsible for chlorpromazine-sensitivity and decreased virulence against silkworm larvae

I speculated that mutants for membrane-associated proteins might be obtained if I selected chlorpromazine-sensitive （CPZs） strains, as CPZ is a drug perturbing membrane fluidity. I isolated one hundred mutants by means of ethylmethane sulfonate treatment. These mutant cells grow under normal conditions of culture but not in presence of CPZ. Out of one hundred, sixteen strains displayed a decreased killing ability compared to the parent strain, in a fifth instar silkworm larvae-infection assay. Then the wild type genes that complement CPZs phenotype were screened from whole genomic DNA library of S. aureus. I present here, one strain named ZS0270 out of three strains that were complemented for CPZs phenotypes. A plasmid pSA0614-SA0615 harboring the genes SA0614 and SA0615 but not a vector pKE515 plasmid complemented CPZs phenotype （Table.1）. In silkworm infection assay, parent RN4220 killed the infected silkworm within 20h whereas ZS0270 took 60h, suggesting the decreased virulence of ZS0270 （Fig.1）. The plasmid also complemented the decreased virulence of ZS0270. DNA sequence analysis revealed that ZS0270 harbored a single amino acid substitution mutation in the gene locus SA0614,Gly59Glu. To confirm the responsibility of the mutation for the CPZs and the decreased virulence, phage transduction experiments were carried out. The results demonstrated that both phenotypes were co-transduced with a certain frequency with a drug-resistant marker inserted near to the SA0614 gene. Therefore, I concluded that the SA0614 gene confers tolerance to CPZ on S. aureus and contributes to S. aureus virulence against silkworm larvae.

A two-component system SA0614 - SA0615 involved in S. aureus tolerance to CPZ and contributing to virulence

The genome database of S. aureus suggested that the SA0614 gene encodes a histidine kinase-activated transcriptional regulator of a two-component system and SA0615 encodes a cognate membrane-integrated histidine-kinase, whose function has not been reported. The insertional disruptants of each gene were constructed, tested for sensitivity to CPZ and decreased virulence. The mutants were unable to grow at a CPZ concentration over 0.08 mM while parent strain grew stably until CPZ concentration 0.15 mM （Fig2A）. The virulence assay on Fig.2B shows that killing of silkworms infected by the deletion mutants was 3 times delayed compared to parent RN4220. These results suggest that the two-component system is responsible for tolerance to CPZ and virulence.

The two-component system SA0614- SA0615 functions to cell wall integrity

The sensitivity of the insertional disruptants to CPZ can be explained by that （i） the two-component system function in membrane homeostasis upon CPZ-induced damage, or （ii） bacterial membrane became more exposed to CPZ because of an altered cell wall structure. To verify these hypotheses, the insertional disruptants were tested for sensitivity to membrane damaging agents such as ionic and non-ionic detergents and cell wall damaging agents. The mutants were strikingly sensitive to the non-ionic detergent including Triton-X 100 （fig.3A） and NP-40 but not to ionic detergents SDS or deoxycholate. The mutants were also more sensitive to lysozyme, a cell wall degrading agent, compared to parent strain RN4220 （fig.3B）. In these cases, non-ionic detergents and CPZ killed the bacteria near micelle-forming concentration whereas ionic detergents killed the bacteria at less than micelle-forming concentration. In all, these results suggest that the cell wall may be altered, resulting to easier exposure of the membrane to the drugs. To confirm the altered cell wall integrity in the by disruption of the SA0614-SA0615 genes, I determined the amount of cell wall peptidoglycan in the mutants and observed a decrease in peptidoglycan amount in the mutant strains compared to parent （Fig.4）. To ask further whether the peptidoglycan alteration resulted from an increased autolysis activity, I performed a zymography analysis of the autolysins of the cell wall of the mutants to determine any increase in activity. The bacteriolytic enzyme profiles that the activity of autolysins was not increased, suggesting that the decreased amount of peptidoglycan may not be caused by an increased synthesis of autolysins.

Discussion:

In this study, I showed that the novel two-component system formed by SA0614-SA0615 contributes to the production of sufficient amount of bacterial cell wall peptidoglycan but also for virulence. The sufficient amount of cell wall peptidoglycan might allow S. aureus to cover the membrane appropriately and to resist cell wall damaging components. This finding suggests that the thickness of cell wall peptidoglycan of Gram-positive bacteria contributes to their pathogenesis, probably by preventing the cell wall and membrane from host attacks. Publication:

Razanajatovo, I.M., Kurokawa, K., Kaito, C., Matsuo, M., and Sekimizu, K. A Novel Two-component System Contributing to Staphylococcal Virulence and Cell Wall integrity. In preparation.

Table.1. Transformation of ZS0270 with a multi-copyplasmid pSA0614-0615 rescued the mutant strain fromCPZ-sensitivity

Fig.4.Decreased of cell wall Peptidoglycan amount from S. aureus mutants .SA0614, .SA0615 but not parent

Fig.5. Perspective of this work

本論文においては、動物感染モデルを用い、病原性細菌の新たな病原性遺伝子を同定した。同定した遺伝子は、新規な二成分制御系で細胞壁の完全性に寄与することを明らかにし、カイコ幼虫感染モデルにおける細菌細胞壁成分の病原性への寄与を考察した。

第一章においては、病原性細菌として黄色ブドウ球菌を用い、変異剤処理により得た変異株をカイコ幼虫感染モデルにおける殺傷能の低下によってスクリーニングを行った。変異剤処理により誘導される点変異を動物感染モデルにおける殺傷能低下という表現型を用いて同定することは困難であるが、申請者は予めクロルプロマジンという膜作用性薬剤に対する感受性株として一次スクリーニングを行い、これを出発材料として病原性低下株を選ぶことによって解決している。即ち、クロルプロマジン感受性と病原性の低下について、同じ変異に起因するものがあると想定し、クロルプロマジン感受性を誘起している変異をプラスミド相補実験により同定し、この変異が同時に病原性の低下を導いていることを変異株一株について明らかにした。

第一章においてはさらに、同定した遺伝子が二成分制御系をコードしていたことを受けて、オペロンをコードする二つの遺伝子の欠損株をそれぞれ作出し、これらの欠損株が先に得た点変異体と同じく、クロルプロマジン感受性と病原性の低下という表現型を示すことを明らかにし、この二成分制御系の病原性への寄与を確かにした。

第二章においては、二成分制御系の病原性への関与について、具体的な分子機能の解明を試みた。初めに毒素を含む病原性因子の発現量に影響を与えているかについて試験したが、検出される主要な毒素に発現量の低下は見られなかった。そこで、宿主免疫からの防御に欠損がある可能性を考えて、リゾチームや抗菌ペプチド、界面活性剤への感受性を調べた。その結果、変異株は細胞壁水解酵素であるリゾチームに感受性であり、またイオン性界面活性剤には感受性を示さないもののTriton X-100などの非イオン性界面活性剤に感受性を示すことを見出した。非イオン性界面活性剤が菌の増殖阻害作用を示すのは、およそミセル形成濃度付近であった。そこで申請者は、界面活性剤の細胞膜への接着を妨げる障壁としての細胞壁の量や構造に変異株では欠損があると推察した。クロルプロマジンもミセル形成濃度付近で細胞増殖の抑制効果を示すことから、クロルプロマジン感受性も同様に説明されると考えられた。

第三章においては、変異株において細胞壁ペプチドグリカンの量が低下していることを見出した。電子顕微鏡での細胞壁の観察では、変異株の細胞壁では電子密度が低下しており、細胞壁の構成成分の密度が低下していることが考えられた。以上の結果は、申請者が見出した二成分制御系が細胞壁の完全性に寄与することを示唆している。

近年の自然免疫の誘導機構の研究からは細胞壁のペプチドグリカンを認識するレセプター蛋白を宿主側が有し、これにより自然免疫の誘導が誘起されることが明らかにされつつある。申請者は多量の黄色ブドウ球菌の接種によりカイコ幼虫の感染死を生じさせていることから、この二成分制御系はおそらくはペプチドグリカンの量と密度の維持を介してカイコ幼虫の自然免疫系の過剰な誘導に関与し、速やかな感染死に寄与することを提言した。

以上の成果は病原性細菌の生理と病原性の解明に貢献するところが大きく、博士（薬学）に値すると判断した。