Klebsiella pneumoniae is an important cause of multidrug‐resistant infections worldwide. Recent studies highlight the emergence of multidrug‐resistant K. pneumoniae strains which show resistance to colistin, a last‐line antibiotic, arising from mutational inactivation of the mgrB regulatory gene. However, the precise molecular resistance mechanisms of mgrB‐associated colistin resistance and its impact on virulence remain unclear. Here, we constructed an mgrB gene K. pneumoniae mutant and performed characterisation of its lipid A structure, polymyxin and antimicrobial peptide resistance, virulence and inflammatory responses upon infection. Our data reveal that mgrB mutation induces PhoPQ‐governed lipid A remodelling which confers not only resistance to polymyxins, but also enhances K. pneumoniae virulence by decreasing antimicrobial peptide susceptibility and attenuating early host defence response activation. Overall, our findings have important implications for patient management and antimicrobial stewardship, while also stressing antibiotic resistance development is not inexorably linked with subdued bacterial fitness and virulence.

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc3Man9GlcNAc2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc3Man9GlcNAc2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at ∼4 S. Man7GlcNAc2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25 °C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity ∼5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447−450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase.

SummaryKlebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia.

Membrane transporter proteins are essential for the maintenance of cellular ion homeostasis. In the secretory pathway, the P-type ATPase family of transporters is found in every compartment and the plasma membrane. Here, we report the identification of COD1/SPF1 (control of HMGCoA reductase degradation/SPF1) through genetic strategies intended to uncover genes involved in protein maturation and endoplasmic reticulum (ER)-associated degradation (ERAD), a quality control pathway that rids misfolded proteins. Cod1p is a putative ER P-type ATPase whose expression is regulated by the unfolded protein response, a stress-inducible pathway used to monitor and maintain ER homeostasis. COD1 mutants activate the unfolded protein response and are defective in a variety of functions apart from ERAD, which further support a homeostatic role. COD1 mutants display phenotypes similar to strains lacking Pmr1p, a Ca 2ϩ /Mn 2ϩ pump that resides in the medial-Golgi. Because of its localization, the previously reported role of PMR1 in ERAD was somewhat enigmatic. A clue to their respective roles came from observations that the two genes are not generally required for ERAD. We show that the specificity is rooted in a requirement for both genes in protein-linked oligosaccharide trimming, a requisite ER modification in the degradation of some misfolded glycoproteins. Furthermore, Cod1p, like Pmr1p, is also needed for the outer chain modification of carbohydrates in the Golgi apparatus despite its ER localization. In strains deleted of both genes, these activities are nearly abolished. The presence of either protein alone, however, can support partial function for both compartments. Taken together, our results reveal an interdependent relationship between two P-type ATPases to maintain homeostasis of the organelles where they reside. INTRODUCTIONDuring biosynthesis, nascent secretory proteins first pass the membranes of the endoplasmic reticulum (ER) through a proteinaceous pore called the translocon (Johnson and van Waes, 1999). In the lumen, ER chaperones and folding catalysts assist their folding and assembly. Because these factors are found only in the ER, the folding state of proteins is monitored to assure that only properly folded proteins traffic to their sites of function. This mechanism, termed "ER quality control," also functions to select irreversibly damaged proteins for degradation. In this mode, misfolded proteins are exported back to the cytosol, presumably through the translocon, where they are ubiquitinated and degraded by the 26S proteasome (for review, see Brodsky and McCracken, 1999;Ellgaard and Helenius, 2001). Other than the terminal step, termed ER-associated degradation (ERAD), the mechanisms governing ER quality control remain poorly understood.Two pioneering studies used genetic methodologies to unravel the mechanisms underlying the degradation of proteins in the ER. One used the direct approach of screening for mutants defective in the degradation of two model misfolded soluble proteins: mutant carboxypeptidas...

We consider configuration of wireless sensor networks, where certain functions must be automatically assigned to sensor nodes, such that the properties of a sensor node (e.g., remaining energy, network neighbors) match the requirements of the assigned function. Essentially, sensor nodes take on certain roles in the network as a result of configuration. To help developers with such configuration tasks for a variety of applications, we propose generic role assignment as a programming abstraction, where roles and rules for their assignment can be easily specified using a configuration language. We present such a role specification language and distributed algorithms for role assignment according to such specifications. We evaluate our approach and show that efficient and robust generic role assignment is practically feasible for wireless sensor networks.

Protein N-glycosylation requires flipping of the glycolipid Man(5)GlcNAc(2)-diphosphate dolichol (Man(5)GlcNAc(2)-PP-Dol) across the endoplasmic reticulum (ER). Helenius et al. report genetic evidence suggesting that Rft1, an essential ER membrane protein in yeast, is required directly to translocate Man(5)GlcNAc(2)-PP-Dol. We now show that a specific ER protein(s), but not Rft1, is required to flip Man(5)GlcNAc(2)-PP-Dol in reconstituted vesicles. Rft1 may have a critical accessory role in translocating Man(5)GlcNAc(2)-PP-Dol in vivo, but the Man(5)GlcNAc(2)-PP-Dol flippase itself remains to be identified.

Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinoneoxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the ␤-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 1.1.1.38 and EC 1.1.1.40, respectively) and of the phosphoenolpyruvate synthase (EC 2.7.9.2) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.In Escherichia coli, several enzymes or pathways are able to convert malate to oxaloacetate. The NAD-dependent (cytoplasmic) malate dehydrogenase (MDH; EC 1.1.1.37) has always been considered to be the principal malate-oxidizing enzyme in the citric acid cycle (tricarboxylic acid [TCA] cycle) of this organism (9). Recently, a malate:quinone-oxidoreductase (MQO; EC 1.1.99.16) which is an essential enzyme in the TCA cycle of Corynebacterium glutamicum (reference 23 and accompanying paper) was described. MQO is a flavin adenine dinucleotide (FAD)-and lipid-dependent peripheral membrane protein catalyzing the oxidation of L-malate to oxaloacetate. The electrons are donated to the electron transfer chain at the level of quinones. This reaction is essentially irreversible (17). In C. glutamicum, MQO and the cytoplasmic NAD-dependent malate dehydrogenase, which is capable of reversible oxidation of malate to oxaloacetate, are active at the sam...