Pathogens of the Streptococcus genus inhabit many different environmental niches during the course of an infection in a human host and the bacteria must adjust their metabolism according to available nutrients. Despite their lack of the citric-acid cycle, some streptococci proliferate in niches devoid of a readily available carbohydrate source. Instead they rely on carbohydrate scavenging for energy acquisition, which are obtained from the host. Here we discover a two-component system (TCS07) of Streptococcus pneumoniae that responds to glycoconjugated structures on proteins present on the host cells. Using next-generation RNA sequencing we find that the uncharacterized TCS07 regulon encodes proteins important for host-glycan processing and transporters of the released glycans, as well as intracellular carbohydrate catabolizing enzymes. We find that a functional TCS07 allele is required for growth on the glycoconjugated model protein fetuin. Consistently, we see a TCS07dependent activation of the glycan degradation pathway. Thus, we pinpoint the molecular constituents responsible for sensing host derived glycans and link this to the induction of the proteins necessary for glycan degradation. Furthermore, we connect the TCS07 regulon to virulence in a mouse model, thereby establishing that host-derived glycan-metabolism is important for infection in vivo. Finally, a comparative phylogenomic analysis of strains from the Streptococcus genus reveal that TCS07 and most of its regulon is specifically conserved in species that utilize host-glycans for growth.

Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites.KEYWORDS Cfr, L3 mutations, antibiotic resistance, linezolid resistance, tiamulin resistance O ver time, more and more mutations in bacterial ribosomal protein L3 (renamed uL3 in accordance with the new universal naming of ribosomal proteins [1]) have been associated with resistance to the antibiotics linezolid (LZD, an oxazolidinone) and tiamulin (TIA, a pleuromutilin). These drugs have overlapping binding sites at the peptidyl transferase center (PTC) in the ribosome (Fig. 1). Specific mutations of 23S rRNA and methylation at position 2503 also cause resistance to these antibiotics, as reviewed in references 2-4. The main part of L3 is positioned on the surface of the 50S ribosomal subunit, but a branched loop extends close to the PTC (Fig. 1), the binding site for various ribosomal antibiotics. The first L3 resistance mutation reported in bacteria was from Escherichia coli selected with TIA, and its role in resistance was verified by genetic evidence (5). Since then, L3 mutations have been associated with resistance to LZD, TIA/valnemulin, and anisomycin, as reviewed in references 2 and 6. Relationships between L3 mutations and antibiotic resistance have been reported in Brachyspira, Staphylococcus, E. coli, and Mycobacterium tuberculosis, but many of the findings lack genetic verification. Our recent study (6) demonstrated a clear antibiotic