b Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin. During the last 10 years, mutations in the L3 ribosomal protein have been associated with bacterial resistance to linezolid (an oxazolidinone) and tiamulin (a pleuromutilin). These drugs bind to the ribosomal peptidyl transferase center (PTC), where specific 23S rRNA mutations and methylation at 2503 also cause resistance to oxazolidinones and pleuromutilins (reviewed in references 1, 2, and 3). 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 many different ribosomal antibiotics. The first L3 resistance mutation in bacteria was detected in 2003 in Escherichia coli selected with tiamulin, and its role in resistance was verified by genetic evidence (4). Since then, L3 mutations have been associated with resistance to linezolid, tiamulin or valnemulin, and anisomycin, as reviewed in reference 1. L3 mutations related to antibiotic resistance have been reported in Brachyspira spp., Staphylococcus spp., E. coli, and Mycobacterium tuberculosis. Most of the studies have lacked proof that these mutations actually cause antibiotic resistance, except a few (4-7). It is not evident exactly which L3 mutations have a relation to reduced antibiotic susceptibility, but only those in the part of the L3 protein nearest to the PTC are assumed to confer a resistance effect. Most of the known L3 mutations are found together with other resistance determinants, mainly mutations in the domain V region of 23S rRNA or mutations in ribosomal protein L4 and/or th...

Abstract The fine balance between proliferation, differentiation, and apoptosis in the colonic epithelium is tightly controlled by the interplay between WNT, Notch, and bone morphogenetic protein (BMP) signaling. How these complex networks coordinate the colonic homeostasis, especially if cancer predisposing mutations such as mutations in the DNA mismatch repair (MMR) are present, is unclear. Inactivation of the MMR system has long been linked to colorectal cancer; however, little is known about its role in the regulation of the colonic homeostasis. It has been shown that loss of MMR promotes the proliferation of colon epithelial cells that renders them highly susceptible to transformation. The mechanism through which MMR mediates this effect, yet, remains to be determined. Using an MMR-deficient mouse model, we show that increased methylation of Dickkopf1 impacts its expression, and consequently, the ability to negatively regulate WNT signaling. As a result, excessive levels of active β-catenin promote strong crypt progenitor-like phenotype and abnormal proliferation. Under these settings, the development and function of the goblet cells are affected. MMR-deficient mice have fewer goblet cells with enlarged mucin-loaded vesicles. We further show that MMR inactivation impacts the WNT–BMP signaling crosstalk.

No abstract

No abstract