Genome‐wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae . However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn‐seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high‐content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi‐based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp‐proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP‐dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.

Polysaccharide capsule is the main virulence factor ofK.pneumoniae, a major pathogen of bloodstream infections in humans. While more than 80 capsular serotypes have been identified inK.pneumoniae, only several serotypes are frequently identified in invasive infections. It is documented that the capsule enhances bacterial resistance to phagocytosis, antimicrobial peptides and complement deposition underin vitroconditions. However, the precise role of the capsule in the process ofK.pneumoniaebloodstream infections remains to be elucidated. Here we show that the capsule promotesK.pneumoniaesurvival in the bloodstream by protecting bacteria from being captured by liver resident macrophage Kupffer cells (KCs). Our real-timein vivoimaging revealed that blood-borne acapsularK.pneumoniaemutant is rapidly captured and killed by KCs in the liver sinusoids of mice, whereas, to various extents, encapsulated strains bypass the anti-bacterial machinery in a serotype-dependent manner. Using capsule switched strains, we show that certain high-virulence (HV) capsular serotypes completely block KC’s capture, whereas the low-virulence (LV) counterparts confer partial protection against KC’s capture. Moreover, KC’s capture of the LVK.pneumoniaecould bein vivoneutralized by free capsular polysaccharides of homologous but not heterologous serotypes, indicating that KCs specifically recognize the LV capsules. Finally, immunization with inactivatedK.pneumoniaeenables KCs to capture the HVK.pneumoniae. Together, our findings have uncovered that KCs are the major target cells ofK.pneumoniaecapsule to promote bacterial survival and virulence, which can be reversed by vaccination.

DNA methylation is an important epigenetic mechanism for phenotypic diversification in all forms of life. We previously described remarkable cell-to-cell heterogeneity in epigenetic pattern within a clonal population of Streptococcus pneumoniae, a leading human pathogen. We here report that the epigenetic diversity is caused by extensive DNA inversions among hsdS_A, hsdS_B, and hsdS_C, three methyltransferase hsdS genes in the Spn556II type-I restriction modification (R-M) locus. Because hsdS_A encodes the sequence recognition subunit of this type-I R-M DNA methyltransferase, these site-specific recombinations generate pneumococcal cells with variable HsdS_A alleles and thereby diverse genome methylation patterns. Most importantly, the DNA methylation pattern specified by the HsdS_A1 allele leads to the formation of opaque colonies, whereas the pneumococci lacking HsdS_A1 produce transparent colonies. Furthermore, this HsdS_A1-dependent phase variation requires intact DNA methylase activity encoded by hsdM in the Spn556II (renamed colony opacity determinant or cod) locus. Thus, the DNA inversion-driven ON/OFF switch of the hsdS_A1 allele in the cod locus and resulting epigenetic switch dictate the phase variation between the opaque and transparent phenotypes. Phase variation has been well documented for its importance in pneumococcal carriage and invasive infection, but its molecular basis remains unclear. Our work has discovered a novel epigenetic cause for this significant pathobiology phenomenon in S. pneumoniae. Lastly, our findings broadly represents a significant advancement in our understanding of bacterial R-M systems and their potential in shaping epigenetic and phenotypic diversity of the prokaryotic organisms because similar site-specific recombination systems widely exist in many archaeal and bacterial species. DNA methylation is a well-known epigenetic mechanism for phenotypic diversification in all forms of life. This study reports our discovery that the Spn556II type-I RM locus in human pathogen Streptococcus pneumoniae undergoes extensive DNA inversions among three homologous DNA methyltransferase genes. These site-specific recombinations generate subpopulations of progeny cells with dramatic epigenetic and phenotypic differences. This is exemplified by the striking differences in colony morphology among the pneumococcal variants that carried different allelic variants of the methyltransferase genes. Phase variation has been well documented for its importance in pneumococcal pathogenesis, but it is currently unknown how this phenotypic switch occurs at the molecular level. This work has thus discovered an epigenetic cause for pneumococcal phase variation. Our findings have a broad implication on the epigenetic and phenotypic diversification in prokaryotic organisms because similar DNA rearrangement systems also exist in many archaeal and bacterial species.

Pathogenic bacteria display various levels of host specificity or tropism. While many bacteria can infect a wide range of hosts, certain bacteria have strict host selectivity for humans as obligate...

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375–Asp-658) of LytB (termed LytB_CAT), excluding the choline binding domain. LytB_CAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a “T-shaped” pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases.

Many encapsulated bacteria use capsules to cause invasive diseases. However, it remains largely unknown how the capsules enhance bacterial virulence under in vivo infection conditions. Here we show that the capsules primarily target the liver to enhance bacterial survival at the onset of blood-borne infections. In a mouse sepsis model, the capsules enabled human pathogens Streptococcus pneumoniae and Escherichia coli to circumvent the recognition of liver-resident macrophage Kupffer cells (KCs) in a capsular serotype-dependent manner. In contrast to effective capture of acapsular bacteria by KCs, the encapsulated bacteria are partially (low-virulence types) or completely (high-virulence types) “untouchable” for KCs. We finally identified the asialoglycoprotein receptor (ASGR) as the first known capsule receptor on KCs to recognize the low-virulence serotype-7F and -14 pneumococcal capsules. Our data identify the molecular interplay between the capsules and KCs as a master controller of the fate and virulence of encapsulated bacteria, and suggest that the interplay is targetable for therapeutic control of septic infections.