Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called “fratricins” such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells’ DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches could be potentially transported to other Gram-positive bacteria. Importance Streptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing over a million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts in identifying new vaccine candidates. Here, we introduced an easy to use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The here used approaches will advance genome editing projects in this important human pathogen.

13CRISPR systems provide bacteria and archaea with adaptive immunity against viruses and 14 plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system 15 can be exploited as a biotechnological tool for precise genome editing at a targeted locus. 16Here, we developed a novel, replicative plasmid that carries the CRISPR-Cas9 system for 17 RNA-programmable, genome editing by counterselection in the opportunistic human 18 pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making 19 targeted, marker-less gene knockouts and large genome deletions. After a precise double-20 stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed 21 repair (HDR) pathway is being exploited to select successful transformants. This is achieved 22 through the transformation of a template DNA fragment that will recombine in the genome and 23 eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered 24 strain, can be easily cured from the plasmid that is temperature-sensitive for replication, by 25 growing it at the non-permissive temperature. This allows for consecutive rounds of genome 26 editing. Using this system, we engineered a strain with three major virulence factors deleted. 27The here developed approaches should be readily transportable to other Gram-positive 28 bacteria. 29 30 31 65 crRNA that is base-paired to a trans-activating crRNA (tracrRNA) forms a two-RNA structure 66 that directs the CRISPR-associated proteins (e.g. Cas9) to introduce a double-stranded break 67 4 (DSB) into the target DNA locus. Site-specific cleavage occurs at locations determined by both 68 base-pairing complementarity between the crRNA and the target protospacer DNA and a short 69 protospacer adjacent motif (PAM) (Jinek et al. 2012). It has been demonstrated that the 70 endonuclease can be programmed by engineering the mature dual-tracrRNA: crRNA as a 71 single RNA chimera (sgRNA for single guide RNA), to cleave specific DNA sites. Thereby, 72 modified versions of the system can be exploited as a biotechnological tool for precise, RNA-73 programmable genome targeting and editing (Jinek et al. 2012). 74After the DSB has been introduced, the cell can utilize two major pathways in order to 75 repair the break and survive: homologous recombination (HR) or non-homologous end-joining 76 (NHEJ). In HR, a second intact copy of the broken chromosome segment, homologous to the 77 DSB site, serves as a template for DNA synthesis across the break. In this mechanism, the 78 crucial process of locating and recombining the homologous sequence is performed by RecA 79 (Shuman and Glickman 2007). NHEJ does not rely on a homologous DNA template, as the 80 two DNA ends are rejoined directly together. Most bacteria such as S. pneumoniae cannot 81 perform NHEJ, while it is capable to perform HR (Prudhomme et al. 2002; 2014). DSB repair 82 can be used as a way to generate mutants or desired changes to the genome by providing a 83 HR template, and forms ...

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. Competent bacteria produce so called fratricins such as CbpD, that kill non-competent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall- and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.