Genomic Profiling Reveals Distinct Routes To Complement Resistance in Klebsiella pneumoniae.

The serum complement system is a first line of defense against bacterial invaders. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. pneumoniae infections. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. More than 90 genes contributed to resistance in one or more strains, but only three, rfaH, lpp, and arnD, were common to all four strains. Deletion of the antiterminator rfaH, which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue; its deletion led to modest reductions in complement resistance. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Our findings show that, even among this small selection of isolates, K. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack

Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes.

Published versio

Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance

Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus

Synergy between Ursolic and Oleanolic Acids from Vitellaria paradoxa Leaf Extract and β-Lactams against Methicillin-Resistant Staphylococcus aureus: In Vitro and In Vivo Activity and Underlying Mechanisms

Combining antibiotics with resistance reversing agents is a key strategy to overcome bacterial resistance. Upon screening antimicrobial activities of plants used in traditional medicine, we found that a leaf dichloromethane extract from the shea butter tree (Vitellaria paradoxa) had antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with further evidence of synergy when combined with β-lactams. Using HPLC-MS, we identified ursolic (UA) and oleanolic acids (OA) in leaves and twigs of this species, and quantified them by HPLC-UV as the major constituents in leaf extracts (21% and 6% respectively). Both pure triterpenic acids showed antimicrobial activity against reference and clinical strains of MRSA, with MICs ranging from 8–16 mg/L for UA to 32–128 mg/L for OA. They were highly synergistic with β-lactams (ampicillin and oxacillin) at subMIC concentrations. Reversion of MRSA phenotype was attributed to their capacity to delocalize PBP2 from the septal division site, as observed by fluorescence microscopy, and to disturb thereby peptidoglycan synthesis. Moreover, both compounds also inhibited β-lactamases activity of living bacteria (as assessed by inhibition of nitrocefin hydrolysis), but not in bacterial lysates, suggesting an indirect mechanism for this inhibition. In a murine model of subcutaneous MRSA infection, local administration of UA was synergistic with nafcillin to reduce lesion size and inflammatory cytokine (IL-1β) production. Thus, these data highlight the potential interest of triterpenic acids as resistance reversing agents in combination with β-lactams against MRSA

Reassessment of the distinctive geometry of Staphylococcus aureus cell division

Staphylococcus aureus is thought to divide in three alternating orthogonal planes over three consecutive divisions. Here the authors dispel this idea, showing that one out of the multiple planes perpendicular to the septum can be used in daughter cells irrespective of its orientation in relation to the penultimate division plane

Phylogenetic distribution of peptidoglycan synthesis enzymes across selected bacterial species.

<p><b>(A)</b> The blue, stacked bars represent the total number of proteins present in each species that have a transpeptidase (dark blue) or transglycosylase (light blue) domain. The total number of proteins that have at least one of the two domains is displayed numerically. Strains highlighted in pink are bacteria reported not to possess cell wall, although they may produce small but functional amounts of peptidoglycan. The maximum likelihood species tree was calculated using PhyML [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004891#ppat.1004891.ref053" target="_blank">53</a>] and concatenated bacterial marker genes identified with the AMPHORA2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004891#ppat.1004891.ref052" target="_blank">52</a>] software. <b>(B)</b> Table showing the peptidoglycan synthesis proteins from <i>S</i>. <i>aureus</i> and their established or hypothetical activities.</p

<i>S</i>. <i>aureus</i> COL MIN showed attenuated virulence in a <i>Drosophila</i> infection model and increased susceptibility to lysozyme.

<p><b>(A, B)</b> Estimated survival curves for wild type (WT) and PGRP-SA mutant (<i>seml</i>) flies infected with COL and COL MIN <i>S</i>. <i>aureus</i> strains or PBS (to monitor the physical effects of the injection <i>per se</i>). WT flies strongly succumbed to infection with COL by 96 hours whereas 88% of WT flies infected with COL MIN survived. Curves were statistically separable, log-rank test P<0.05. At least 90% of the PGRP-SA-deficient flies were killed by WT bacteria (within 60 hours) and by COL MIN mutant strain (within 96 hours). Curves were statistically separable, log-rank test P<0.05. (<b>C)</b> Bacterial cell lysis monitored through the decrease of OD₆₀₀ was determined for COL and COL MIN strains in the presence (+) or absence (-) of lysozyme (300 μg/ml). The minimal strain showed increased cell lysis in the presence of lysozyme. Data shows mean with 95% confidence intervals of three independent biological repeats.</p

<i>S</i>. <i>aureus</i> minimal mutant strain COL MIN displays normal growth and requires PBP1 and PBP2 for survival.

<p><b>(A)</b> Growth of the parental strain COL and the minimal mutant strain COL MIN was followed in rich liquid medium by monitoring the absorbance at OD_600nm. The mutant strain COL MIN (doubling time 40 min) showed similar growth to the parental strain COL (doubling time 36 min). <b>(B)</b> Growth of COL and COL MIN was followed in minimal medium by monitoring the absorbance at OD_600nm. The mutant strain COL MIN (doubling time 67 min) showed similar growth to the parental strain COL (doubling time 61 min). <b>(C)</b> Depletion of PBP1 from COL PBP1i and COL MIN PBP1i, in which PBP1 expression is under the control of the IPTG inducible P_<i>spac</i> promoter, by growing cells in the absence of IPTG, led to a halt in cell growth and subsequent drop in optical density indicating PBP1 is essential for survival of both the parental and mutant strains. <b>(D)</b> In the absence of PBP2, strain COL PBP2i (parental strain COL with PBP2 expression under the control of the IPTG inducible P_<i>spac</i> promoter) continues to grow. However, depletion of PBP2 from COL MIN PBP2i causes arrest in growth indicating PBP2 is essential for growth of COL MIN. Averages of three independent replicates are shown and error bars show standard deviations.</p