How bacteria survive an acid trip.

نویسندگان

  • Karan S Hingorani
  • Lila M Gierasch
چکیده

The evolutionary pressure to populate rewarding niches can require organisms to survive high-risk environments. For bacteria that inhabit the nutrient-rich human gut, whether they are our friends or foes, the trip through the stomach requires clever strategies to survive harsh, low-pH conditions. Gut-resident Escherichia coli strains deploy a complex set of responses to counter the impact of the low pH they experience as they travel through the stomach (1). Some of their responses, such as the amino acid decarboxylases, act to keep the cytoplasmic pH above a dangerous level. However, the permeability of the outer membrane leaves the periplasmic space unprotected from the perilously low pH of the external medium. Consequently, these bacteria had to develop strategies to protect periplasmic proteins from irreversible pH denaturation. A network of chaperones participates in this protection; the key pH-responsive members are the small, abundant HdeA and HdeB proteins, the activity of which is triggered by low pH. In a recent study reported in PNAS, Foit et al. (2) apply a multipronged approach to learn how E. coli HdeA uses low pH-induced protonation of a small number of acidic residues to shift from an inactive, stably folded dimeric state to a partially folded monomer that is capable of reversibly binding unfolded substrates. The mystery underlying HdeA function is how pH could change its properties in such a way that turns on its chaperone activity. Given the pH shift that this protein would experience between the stomach (pH 2) and the small intestine (pH 7), the most likely titratable residues involved in the modulation of activity are aspartate or glutamate. Foit et al. (2) identify several potential “pH switches” based on conservation of Asp and Glu residues in the HdeA family, the location of the conserved Asp and Glu residues on the structure of the inactive dimeric form of HdeA, and a powerful computational method called constant pH molecular dynamics (CpHmd) calculations (3). The CpHmd calculations are particularly informative, because they provide estimates of the pKa shifts each Asp and Glu residue would experience between the low-pH stable dimer (4) and models of the monomeric active state of HdeA built from the dimer. Strikingly, the authors find that mutation of the two aspartates predicted to experience the largest pKa shifts (D20 and D51), which were also among the most highly conserved acidic residues, to alanines created a variant HdeA that is constitutively active at neutral pH. Using thermodynamic coupling relationships, the pKa shifts were related to the expected extent of destabilization of the dimer by the Asp to Ala mutations, and, indeed, the two resulting HdeA variants showed a substantial reduction in apparent dimer-melting temperature. Mutation of other acidic residues, which were not predicted to shift pKa to as great an extent as D20 and D51, hardly perturbed the apparent dimer melting temperature. Notably, the pH-dependent ability of the D20, D51 HdeA variant to bind a fluorescent dye, used as a measure of the exposure of the hydrophobic surface, was shifted significantly toward higher pH relative to that of wild-type HdeA. Also, circular dichroism showed that the constitutively active double mutant had significantly reduced secondary structure at neutral pH than the wild-type protein, and the loss was similar to that triggered in wild-type HdeA by lowering pH. Most importantly, the double mutant completely blocked aggregation of unfolded malate dehydrogenase at pH 5, and substantially inhibited it at neutral pH, substantiating the identification of D20 and D51 as major pH-switch residues. Analytical ultracentrifugation analysis confirmed that the exposure of hydrophobic surface, loss of secondary structure, and increase in chaperone activity were all coincident with a shift in the wildtype HdeA dimer–monomer equilibrium toward monomer for the D20A, D51A variant. How does HdeA function as a chaperone? Major classes of bacterial cytoplasmic chaperones like GroEL and DnaK use ATP binding and hydrolysis to switch between highand low-affinity states and to set the timing of a cycle of binding and release of unfolded substrates. These mechanistic steps simultaneously optimize folding assistance and minimize accumulation of unfolded substrates, thus lowering the risk of aggregation. In the case of HdeA, which must function in the periplasm where there is no ATP, pH gradients appear to play a role comparable to ATP. Upon exposure to low pH, the HdeA dimer rapidly dissociates, and the monomer binds an array of different acid-denatured, periplasmic proteins (4, 5) (Fig. 1). Among them, intriguingly, are DegP and SurA, themselves periplasmic chaperones. The shift back to higher pH after the bacterium’s traversal of the stomach leads to a relatively slow release of bound clients from HdeA, apparently enabling refolding to occur with minimal risk of aggregation. It will be interesting to determine the rate of release and refolding of the DegP and SurA chaperones to see whether they might be available to facilitate the refolding of other HdeA clients. Similarly, it will be of interest to determine the role of HdeB, which shares many properties with HdeA but, from in vitro studies, has a different optimal pH range for its chaperone function (6). It seems likely that these two similar small periplasmic chaperones act as a synergistic team. The study by Foit et al. (2), combined with previous work from this group (7, 8), makes a compelling case for a direct link between partial unfolding in HdeA and its enhanced chaperone activity. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions of proteins (IDRs) have recently entered the limelight of the protein science world (9, 10). Defying the dogma that 3D structure is required for a protein to function, IDPs and IDRs exist as ensembles of highly dynamic states. IDPs and IDRs are implicated in molecular recognition, signaling, interdomain linkages, etc., and their structural plasticity is implicated in their promiscuous binding (10, 11). Although the molecular details are not yet clear, the fact that HdeA chaperone activity is associated with its partially folded monomeric state suggests a role for conditionally disordered regions in HdeA in binding a wide array of unfolded substrates. These regions

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Survival of Wolbachia pipientis in cell-free medium.

Wolbachia pipientis is an obligate intracellular bacterium found in a wide range of invertebrate taxa. While over ecological timescales Wolbachia infections are maintained by strict maternal inheritance, horizontal transfer events are common over evolutionary time. To be horizontally transferred between organisms, Wolbachia bacteria must pass through and survive an extracellular phase. We used ...

متن کامل

The Contamination Rate of Industrial Fruit Juices Supplied in Kerman City with Gram-Positive Cocci Bacteria in 2015:A Short Report

Background and Objectives: Some bacteria have the ability to survive in juice after pasteurization. The aim of this study was to evaluate the contamination rate of industrial juices with grampositive bacteria via microbial culture. Materials and Methods:  This descriptive study was conducted on 165 samples of 25 different brands of beer. The specimens were incubated in blood agar medium for 48...

متن کامل

An In Vitro Study on Impact of Environmental Stresses on Growth, Morphological and Biochemical Features of Listeria monocytogenes PTCC 1297

Introduction: Listeria monocytogenes is a serious concern for the food industry due to its high case fatality rate, widespread distribution, ability to survive a wide variety of food processing conditions, and the severity of the illness associated with this pathogen infection. The objective of this study was to determine the growth, cell morphology and biochemical characteristics of L. monocyt...

متن کامل

MECHANISMS OF RESISTANCE TO QUINOLONES AND EPIDEMIOLOGICAL SIGNIFICANCE OF Salmonella spp

Bacteria develop resistance to antimicrobial agents by a number of different mechanisms. The resistance to (fluoro)quinolones in Salmonella is of particular importance especially if therapy in humans is required. For decades there has been a significant interest in studying the biology of Salmonella because these bacteria are among the leading causes of foodborne illnesses around the globe. To ...

متن کامل

The survival of silage inoculant lactic acid bacteria in rumen fluid.

AIMS To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and ferment...

متن کامل

On the Race to Starvation: How Do Bacteria Survive High Doses of Antibiotics?

In this issue of Molecular Cell, Gutierrez et al. (2017) unravel a bacterial survival strategy that they term "density-dependent persistence" or DDP. The authors demonstrate that the majority of isogenic cells in bacterial populations survive lethal antibiotic doses once bacteria consume nutrients and enter stationary growth phase.

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Proceedings of the National Academy of Sciences of the United States of America

دوره 110 14  شماره 

صفحات  -

تاریخ انتشار 2013