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Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

机译：肺炎链球菌生物膜突变体的分离及其在鼻咽菌落定殖过程中的鉴定

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Asymptomatic colonization of the nasopharynx by Streptococcus pneumoniae precedes pneumococcal disease, yet pneumococcal colonization factors remain poorly understood. Many bacterial infections involve biofilms which protect bacteria from host defenses and antibiotics. To gain insight into the genetics of biofilm formation by S. pneumoniae, we conducted an in vitro screen for biofilm-altered mutants with the serotype 4 clinical isolate TIGR4. In a first screen of 6,000 mariner transposon mutants, we repeatedly isolated biofilm-overproducing acapsular mutants, suggesting that the capsule was antagonistic to biofilm formation. Therefore, we screened 6,500 additional transposon mutants in an S. pneumoniae acapsular background. Following this approach, we isolated 69 insertions in 49 different genes. The collection of mutants includes genes encoding bona fide and putative choline binding proteins, adhesins, synthases of membrane and cell wall components, extracellular and cell wall proteases, efflux pumps, ABC and PTS transporters, and transcriptional regulators, as well as several conserved and novel hypothetical proteins. Interestingly, while four insertions mapped to rrgA, encoding a subunit of a recently described surface pilus, rrgB and rrgC (encoding the other two pilus subunits) mutants had no biofilm defects, implicating the RrgA adhesin but not the pilus structure per se in biofilm formation. To correlate our findings to the process of colonization, we transferred a set of 29 mutations into the wild-type encapsulated strain and then tested the fitness of the mutants in vivo. Strikingly, we found that 23 of these mutants were impaired for nasopharyngeal colonization, thus establishing a link between biofilm formation and colonization.
机译：肺炎链球菌在无症状的情况下被肺炎链球菌定居于鼻咽，但对肺炎球菌的定居因子仍知之甚少。许多细菌感染都涉及生物膜，可以保护细菌免受宿主防御和抗生素的侵害。了解 S形成生物膜的遗传学。肺炎，我们对具有血清型4临床分离株TIGR4的生物膜改变的突变体进行了体外筛选。在6,000个 转座子突变体的首次筛选中，我们反复分离了生物膜过量产生的荚膜突变体，表明该胶囊对生物膜形成具有拮抗作用。因此，我们在 S中筛选了6,500个其他转座子突变体。肺炎荚膜的背景。按照这种方法，我们在49个不同的基因中分离了69个插入片段。突变体的集合包括编码真正和假定的胆碱结合蛋白，粘附素，膜和细胞壁成分的合酶，细胞外和细胞壁蛋白酶，外排泵，ABC和PTS转运蛋白以及转录调节子的基因，以及一些保守的和新颖的假设的蛋白质。有趣的是，当四个插入映射到 rrgA 时，编码了最近描述的表面菌毛的一个亚基 rrgB 和 rrgC （编码了其他两个菌毛亚基） ）突变体没有生物膜缺陷，与生物膜形成过程中的RrgA粘附素有关，但与菌毛结构无关。为了将我们的发现与定殖过程相关联，我们将一组29个突变转移到野生型封装菌株中，然后在体内测试了这些突变体的适应性。令人惊讶的是，我们发现这些突变体中有23个因鼻咽定植而受损，从而在生物膜形成和定植之间建立了联系。

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