Udaat Mittu

Bacterial Stress Responses A review by: Udaat Mittu- Integrated PhD in Biology Principal Investigator: Dr Sunish Kumar Radhakrishnan 1 Abstract: Antimicrobial resistance is one of the major contributors to deaths worldwide. Recent data from WHO suggests a worrying upwards trend in the lethalities caused by microorganisms which survive and grow in the presence of antibiotics. The plan of action then is to prevent the acquisition of resistance by these organisms so as to save lives. To that effect, what is required is an understanding of how pathogens escape death in the face of antimicrobial stress so that we can devise methods of countering it. Therein lies the reason behind this review. Antimicrobials induce stress upon bacteria and bacteria survive by responding to those stresses. It would serve us well to understand how bacteria sense the presence of stress and react accordingly. A variety of responses are exhibited by bacteria to a variety of stressors, be it temperature, pH, DNA damage etc. So, section by section, we shall try to uncover the canonical stress responses of bacteria and then see how generation of stress responses connects to the ultimate problem of development of antimicrobial resistance. The stress responses: In this section, a brief introduction to the various stress responses of bacteria which are known to date will be provided. Information related to the activation and sensing mechanisms of these stress responses shall be discussed. Effector functions shall be avoided given the brief scope of the review however, in another section, the involvement of some of these responses in development of persistence, tolerance or resistance to antimicrobials shall be tackled. With that, let’s start exploring the stress responses of bacteria. Temperature related stress responses The Heat shock response - The heat shock response is induced by increase in the levels of σ32, a transcription factor coded by rpoH. It is responsible for activation of various heat shock proteins that allow a bacterium to survive conditions of high temperature. Increased temperature causes a transient 15-20 fold increase in the levels of σ32 that stabilises to 2-3 fold higher as compared to a bacterium growing at the optimum temperature. Levels of σ32 are regulated by 3 different mechanisms - (i) By self complementarity of rpoH mRNA which blocks its ribosome binding site (RBS) (ii) by binding to DnaK/DnaJ/GrpE chaperones (iii) by decreasing stability of σ32 at room temperature. (1) (i) At non-stress inducing temperatures, the rpoH mRNA exists in a secondary structure which blocks the RBS of the mRNA. There are two 5’ proximal regions of the mRNA which are responsible for its temperature based regulation called region A and B. These were detected via rpoH-lacZ reporter fusions which were subjected to a deletion analysis where small regions of the rpoH gene were deleted starting from the 3’ end and step by step moving towards the 5’ end. By doing so, it was observed that regions A and B, when deleted, cause rpoH mRNA to become temperature insensitive and express at high levels at 30ºC. A is responsible for enhancement of translation whereas region B is responsible for repression of the mRNA at lower temperatures. These results were verified by introducing point mutations in these regions in an independent study. (2,3) These studies show therefore that the mRNA is a thermosensor and can be induced by heat. The main mechanism being that when temperature increase, the rpoH mRNA melts, hence revealing the RBS. (11) (ii) DnaK-DnaJ-GrpE point mutations showed increase in stability of σ32 at low temperatures hence signifying that this chaperone is closely involved in regulation of σ32. (4) (iii)FtsH was one of the first proteases shown to be involved in causing cleaving of σ32 at room temperature by showing that purified FtsH can degrade σ32 in vitro. It was also shown that a specific region of σ32 is involved in FtsH based regulation by introducing frameshift mutations and deletions to show that σ32 with mutations in this specific region, labelled the ‘RpoH box’, is stable at room temperature. (5,6) It was later found that mutations in this region of σ32 affect its binding to both DnaK and RNAP. Binding to DnaK leads to inhibition while binding to RNAP leads to stabilisation. (7,8) Thus, RNAP and DnaK compete for binding to this region of σ32 and lead to different consequences. When a bacterium experiences increased temperatures, levels of unfolded proteins which can bind to chaperones increases, thus reducing the availability of DnaK chaperones which shifts the equilibrium towards RNAP which promotes stabilisation of σ32. This was proved by showing that at lower temperatures, just by increasing the amount of unfolded protein, a heat shock response could be induced. (9,10) The counterbalancing is performed by cellular proteases such as FtsH which prevent over accumulation of σ32 (12) and in this manner, a two to three fold increase in the levels of σ32 is achieved at higher temperatures. The gram positive bacterium heat shock response is different. It involves HrcA and CIRCE (controlling inverted repeat of chaperone expression). HrcA is repressed by a HrcA repressor which is bound to GroE chaperone. When levels of misfolded proteins increases with increased temperature, amount of GroE which can remain bound to the repressor decreases and as a result, the activity of the repressor decreases and HrcA is activated. (13) Formation of non native proteins activates the CIRCE regulon. (14) Thus, with increase in temperature, HrcA is activated and a heat shock response is produced. The HrcA/CIRCE regulons are present in a diverse range of organisms from Chlamydia and Spirochaeta (15,16) which have both dnaK and groE operons, to ⍺ proteobacteria such as Caulobacter crescentus which has only the groE operon under the control of CIRCE/ HcrA. (16,17) It is also important to mention here that in Caulobacter crescentus, the heat shock response is positively controlled by RpoH and controlled negatively by HcrA. (17,18) 2 Cold shock response - The response occurs on three fronts - (i) membrane associated changes (ii) nucleic acid associated responses (iii) Csp regulon. (I) The membrane response is associated with adjustment of lipid composition of a membrane. Unsaturated fatty acid (UFA) composition increases upon encounter with lower temperatures. UFAs reduce the melting point of the membrane and thus maintain the fluid properties of the lipid bilayer. (19) Points of differences exist between gram negative and gram positive organisms. In Escherichia coli, enzymes such as β-ketoacyl-acyl carrier protein synthase II is expressed at normal temperatures and is activated when the temperature falls. Upon activation it produces cis-11-octadecenic acid (a UFA), using palmitoleic acid as a substrate. (19-21) In contrast, in gram positive organisms such as Bacillus subtilis, the response is cold induced rather than cold activated. Enzyme desaturase is expressed from des gene when the temperature falls. The enzyme inserts a double bond into the acyl chain of membrane phospholipids hence introducing UFAs into the membrane in response to decrease in temperature. (22) (II) Nucleic acids themselves can serve as thermosensors by becoming negatively supercoiled at lower temperatures. (23) In both Escherichia coli and Bacillus subtilis, transient increase in negative supercoiling was observed as temperatures fell. This was established by transforming a plasmid inside Bacillus subtilis, inducing stress on the organism, isolating the plasmid and then running it on a gel to see how the plasmid was altered. It was observed that negative supercoiling increased in response to a cold shock. Further, positive supercoiling increased as a response to heat shock. Nucleic acids also respond by altering their supertwist in response to a variety of stresses besides temperature based stresses. (24) It is obvious that changes in the nature of folding of the nucleic acids will have large scale ramifications. Existence of twist sensitive promoters which respond to such changes supports this idea e.g. recA has a cold shock inducible twist sensitive promoter. (23) (III) Csp (cold shock protein) regulon is spear headed by CspA protein which was identified in Escherichia coli and since then has been found to have homologs in more than 60 species including gram-positive, thermophilic, gram negative, psychotrophic and mesophilic bacteria. (25) The response is immediate and is followed by low temperature adaptation which allows growth at low temperatures. (26) Induction of cold shock leads to a transient growth lag phase during which Csp expression increases dramatically. This was observed in Escherichia coli by performing two dimensional gel electrophoresis for total cellular proteins which were labelled with radioactive methionine. (S35) The protein levels were observed at varying time points after the temperature downshift and it was observed that cold shock proteins are expressed at a higher rate while other cellular protein expression is arrested for a short period, which is followed by cold adaptation where Csp proteins continue to be expressed alongside other cellular proteins. (25) The Csps expressed are divided into two classes, where class I represents proteins induced dramatically upon cold shock and class II represents proteins which are present at 37ºC and are marginally upregulated during cold shock response. (27) The Csp regulon comes under class I (28-30) while proteins such as RecA come under class II. (31) Proteins such as peptidyl prolyl isomerase (better known as a trigger factor) are important for refolding of proteins which are damaged due to cold temperatures. This enzyme is cold induced and its over expression in Escherichia coli leads to increased viability of cells. (32) The common theme of temperature related stress responses is that both responses display transient increased expression followed by temperature adaptive behaviour which allows the bacterium to grow at stress inducing temperatures. Envelope stress response - Envelope stress response is generated by 2 different regulons under the control of two orthogonal factors (I) σE and (II) CpxAR two component system. (I) σE - This response is activated when mutations or events which cause protein misfolding in the envelope or overproduction of mutated envelope proteins occur. Misfolding of outer membrane proteins or mutations in the periplasmic proteins also produces the same response. (33) The signal generated by misfolded proteins is transduced across the inner membrane via a membrane localised factor called RseA. A 25 fold increase in σE pathway activity is observed when null mutations are introduced in rseA gene. After these mutations are made, σE response becomes insensitive to envelope stress and is always active. Overexpression of RseA represses the σE pathway. (34,35) Another member of this is the RseB protein. Null mutations in the rseB gene cause only a two to three fold increase in the σE response and over expression of RseB suppresses the σE response but not completely, thus it serves as a molecule which fine tunes the σE stress response. (36,37) When σE activating signals are received, DegS mediated degradation of RseA occurs which activates the σE pathway completely. (38) (II) CpxAR - system activates when some sort of perturbation to envelope protein biogenesis occurs. Cues which can cause activation of this pathway are altered pH, improper folding of inner membrane proteins or absence of phosphatidyl ethanolamine (PE) in mutants, or perturbation of protein folding and its localisation to the bacterial envelope. (39-41) This signal for activation is transferred across the envelope by the sensor histidine kinase (HK) CpxA and the response regulator (RR) CpxR. (42-44) CpxA functions as an autokinase, CpxR kinase and a phosphatase for the CpXR which has been phosphorylated already. The ratio between CpxA kinase vs the CpxA phosphatase activity determines the extent of activation of this pathway. (43) As CpxR is phosphorylated, it gains the ability to bind to consensus sequences present upstream of the cpxactivated genes and thus activates the expression of 3 the Cpx mediated regulon. (43,45) Another moiety known as CpxP exists which when deleted leads to a constitutive three- to five- fold increase in the activity of the CpxAR pathway. It serves a similar function as the RseB molecule in the σE pathway in that it serves as a method of fine tuning the CpxAR response. (46,47) CpxP can bind to the sensing domain of CpxA and maintenance of a CpxA:CpxP ratio of 1:1 inhibits the auto kinase activity of CpxA by about 50%. (47,48) This ability allows CpxP to serve as a repressor molecule for the CpxAR pathway. SOS response SOS response occurs in response to DNA damage and it leads to the activation of greater than 20 genes. (49) The response consists of two separate components LexA and UmuD. UmuD and LexA share homology in their carboxy-terminal domains. Both of these proteins undergo an autodigestion when they are acted upon by an activated RecA protein which exists on a RecA/ ssDNA nucleoprotein filament where the RecA protein polymerises. This RecA/ssDNA assembly (RecA*) serves to activate components under LexA and UmuD. (50-54) Thus, cellular RecA serves as a sensor for DNA damage and activates the SOS response. (I) LexA - The protein LexA serves as an inhibitor to various components of the SOS response by binding to their similarity regulatory sequence (SOS box) which is present close to the promoters of these genes. The interaction between RecA* and LexA activates the inherent ability of LexA to autodigest. (53,54) The autodigestion occurs between the A84G85 residues (55) thus inactivating the repressor function of the protein and activating all the genes under its repression. RecBCD enzyme functions in the case of a double stranded break in the DNA. This RecBCD complex in tandem with single stranded binding proteins activates RecA downstream in the pathway which leads to the same response. (56) As the level of LexA in the cell decreases due to autodigestion, various genes under the repression of LexA get activated. (II) UmuD - umud and umuc genes exist in an operon which is under the repression of LexA. (57) Like LexA, UmuD gets cleaved by RecA*, although at a slower rate as compared to LexA, but which nonetheless leads to the formation of UmuD’ that along with UmuC allows the pathway to perform its function which is SOS mutagenesis, better known as translesion synthesis. (50,51,58) It is important to mention here that uncleaved UmuD in a complex with UmuC serves an important role in cell cycle regulation. This complex serves as a checkpoint for DNA damage and allows repairs to be completed before the cell can replicate its DNA. (59) UmuD and UmuD’ in their functional complexes exist as homodimers which are linked to each other via Cysteine amino acids present in fixed positions which form a disulphide bond. (60,61) In the UmuD2C or UmuD’2C complex, UmuC itself is a DNA polymerase which can function in translesion synthesis, however, another polymerase can be activated by the UmuDC pathway which is the DinB polymerase. (62-65) dinB gene was found to be necessary for generating untargeted mutations in Escherichia coli.(62,63) DinB is an error prone polymerase which explains why DinB gene is necessary for mutagenesis. (66) It lacks 3’ to 5’ proofreading exonuclease activity, thus it cannot proof read the nucleotides it adds. Further, DinB also has the ability to introduce -1 frameshift mutations which can lead to large scale changes. (63,67) Thus, the SOS response which occurs in response to DNA damage acts to both repair the DNA, perform bet hedging strategies such as using an error prone polymerase to fish for mutations and it also holds influence over the cell cycle such that it can pause the cell cycle to ensure proper repair of DNA before the bacterium proceeds to replicate the DNA. Oxidative stress response Oxidative stress response is regulated by the OxyR and SoxRS regulons. The OxyR locus consists of genes which are induced by H2O2 (68) while the SoxRS locus consists of genes which are induced by the presence of O2•- moeity. (69,70) (I) OxyR - OxyR based sensing occurs via the formation of a disulphide bond between C199 and C208 which occurs due to oxidation of OxyR. This was established via a two pronged approach. Mutation of either of the two cysteine residues led to the development of an OxyR protein which was unable to sense the presence of H2O2 in the medium. Further, a mass spectrometric analysis was performed for both the reduced and oxidised forms of OxyR. Using that data, it was established that the oxidised form of OxyR had a disulphide bond while the reduced OxyR had thiol groups at the cysteine residues. Hence, the mechanism of sensing of H2O2 was deduced. OxyR must be oxidised directly for it to perform its function. (71) It was shown using synthetically designed oligonucleotides which were selected for using electrophoretic mobility shift assays followed by purification and sequencing via Sanger’s dideoxy sequencing method that the oxidised and reduced forms of OxyR bind to different consensus sequences. The consensus sequences for both the oxidised and reduced forms of OxyR were found to be - ATAGnt elements, four of them present on major grooves for oxidised form, while for the reduced form, the sequence was two ATAGnt elements which were on major grooves separated by one helical turn of the DNA. (72) Thus, oxidation of OxyR would lead to its activation and also change its preferred consensus sequence, hence activating the OxyR regulon. (II) SoxR - SoxR recognises the O2•- moeity via iron sulphur clusters.The oxidation of SoxR iron sulphur cluster from [2Fe-2S]1+ to [2Fe-2S]2+ is how it is activated. This mechanism was proved by performing dithionite induced reduction of Fe-SoxR protein and it was shown that the SoxR was able to regain its ability to activate transcription after autoxidation. (73) This finding was bolstered by the fact that a mutant SoxR which was constitutively 4 active was found to exist in its oxidised form even in the absence of any stress. (74) It is after its activation that SoxR can induce the transcription of genes which exist under the SoxRS regulon. OxyR and SoxRS exist in a wide variety of organisms. OxyR homologs have been detected in organisms such as Erwinia carotovora (first oxyR homolog to be found by performing DNA hybridisations), Mycobacterium species, and even Xanthomonas campestris. (75,76,77) SoxR homologs have been detected in Pseudomonas aeruginosa, Bordetella pertussis, Vibrio cholerae and many more bacteria. (78) OxyR and SoxRS perform many more functions besides just providing a defense against reactive oxygen species. OxyR has been shown to provide resistance against HOCl and organic solvents and even some reactive nitrogen species. SoxRS has been deemed important in response to antibiotic related stresses, where some articles have went on to say that SoxRS activation could be a comprehensive response against xenobiotic agents rather than just a defense against O2•-. (78) Stringent response - The stringent response is activated in response to nutritional starvation and a multitude of other stresses such as osmotic stress and even exposure to antimicrobial compounds and xenobiotics. The major player in the stringent response is the alarmone (p)ppGpp which is synthesised by 2 categories of enzymes - (i) RelA/SpoT homologue proteins (RSH) (ii) small alarmone synthetases (SAS) and small alarmone hydrolases (79,80) (I) RSH genes - The RSH gene in Escherichia coli is RelA and it is under the control of 4 promoters. P3 and P4 of σ54 and P1 and P2 of relA itself. The homolog for RelA in gram positive bacterium is a RelA/SpoT enzyme which contains both synthetase and hydrolase domains. (79) RelA is activated by different molecular players under different types of nutrient starvation conditions or even different types of stresses. CRP can activate relA under carbon stress, σ54 can activate P3 and P4 of relA when nitrogen starvation conditions are encountered. HNS can activate relA when heat shock stress is encountered and rpoS, which codes for σS can activate relA when osmotic stresses are encountered. (81-83) (II) SAS genes - SAS genes are differentially regulated during growth. This was discovered when sequence similarity searches were performed in Bacillus subtilis which was believed to have only one RelA/ SpoT homologue. Two genes, named yjbM and ywaC, were identified and after deducing their amino acid sequence, it was found that both of these genes had similarity to RelA/SpoT (p)ppGpp synthetase domains. Northern blot analyses revealed that these genes were differentially expressed during different phases of growth. (84) Multiple genes such as this exist in a variety of organisms. For example, relP exists in Bacillus subtilis which has similar functions. (79) RelP is under the control of σM induced regulon which is responsible for generating a response to envelope stress caused by alkaline shock or by stress induced by the antimicrobial vancomycin. (85) Besides these genes, (p)ppGpp production can be affected by additional control of the alarmone synthesising enzymes themselves. (p)ppGpp synthesising enzymes are known to have differing preferences for their substrate. Some enzymes such as the RelA of Escherichia coli show a greater preference for GDP whereas the Rel homologue of Mycobacterium tuberculosis has a greater preference for GTP. This was found to occur due to presence of either an EXDD or a RXKD domain in the substrate binding domain for these enzymes. By performing a (p)ppGpp synthesis assay with enzymes with each of these domains, it was found that an EXDD domain preferred GDP as a substrate and RXKD was partial towards GTP. (86) In this manner, substrate preferences can serve as a control on the activity of these enzymes. Beyond that, the product (p)ppGpp itself when produced, can promote the transcription of relA as seen in Escherichia coli, thus creating a positive feedback loop. (87) However, even this positive feedback loop can be product selective. It was observed in Bacillus subtilis that pppGpp can positively regulate the rel homologue (RelQ), but ppGpp fails to do so. This occurs because RelQ must form a tetramer with two pppGpp molecules which occurs via the interaction of pppGpp molecules to the allosteric binding site of the enzyme. ppGpp fails to do so. This was revealed by isolating the tetramer and discerning its crystal structure. (88) Thus, (p)ppGpp production is controlled by a variety of mechanisms and it is understandable why it is so given that it is involved in large scale responses to a variety of stresses. These functions shall be discussed in the next section where we will take a look at second messengers in bacteria. General Stress response - General stress response is under two different master regulators in Gram positive and Gram negative bacteria. Gram negative bacteria are under the control of rpoS gene coded σS factor (Escherichia coli), whereas the Gram positive bacteria are under the control of sigB gene coded σB. (Bacillus subtilis) We shall cover in brief the regulation of these factors in this section and their functions in the next section where we look at the master regulators in stress responses. σS in Escherichia coli σS is a master regulator of the stationary phase of growth in Escherichia coli and governs a large number of stationary phase genes. (78) σS controls a different set of genes than σ70 since a deletion of the rpoS gene completely removes the expression of σS induced genes and σ70 is unable to induce the same genes. So, unlike factors like heat shock factor σ32 which accumulates to a certain level and competes against σ70 to change the global expression pattern, σS controls a different set of genes entirely. (89,90) σS is regulated tightly in different stages of growth. Its cellular levels are almost non-existent during the log phase of growth, however, as various stresses start to accumulate the levels of σS start to increase. Thus, conditions encountered during stationary phase of growth such as nutrient starvation, altered pH of the 5 medium or increased cell density tend to induce translation of the rpoS mRNA. (89,91-97) Since this factor responds to so many different kinds of stresses, it is regulated at multiple levels. The major mRNA transcript for σS is generated by rpoSp. The rpoS mRNA is expressed at low levels during log phase of growth, but as the cells move into lag phase, the levels of this mRNA starts to increase. (94,98-101) It was also found that a second messenger cAMP is a repressor of rpoS transcription as both crp and cya loss of function mutations increased the levels of rpoS transcription which was observed using a rpoS::lacZ fusion. (94) Another second messenger ppGpp was found to positively affect rpoS transcription. (92,102) Regulation of rpoS goes beyond just transcription. Suspicions arose regarding this when different reports were received for rpoS::lacZ fusions for transcription and translation. (94,99,103) It was then discovered that the protein Hfq is required for rpoS mRNA translation. A small RNA was also found to be involved in the control of rpoS translation. It turned out to be DsrA which showed upregulation of rpoS translation when lower temperatures were experienced by the bacterium. (104-106) Another level of control of σS is at the post translational level. σS can undergo proteolysis. The half life of σS was estimated to be between 1 to 4 minutes in log phase cells, but as the cells progress into lag phase, the stability of σS increased steadily. (91,94) The proteins which are essential for this are ClpX and RssB. RssB can bind directly to σS and it acts as a chauffeur which presents σS to ClpXP for proteolysis. Phosphorylation of RssB increases the affinity of RssB to σS leading to the conclusion that environmental cues can alter σS proteolysis by modulating the binding of RssB to σS. (91,107,108) Thus, a master regulator like σS is regulated at multiple levels and responds to a large variety of stresses, hence its categorisation as a general stress response. A general rule of thumb for this response is that it is generated when the stress increases gradually as compared to one which can be immediately lethal to the bacterium. Besides Escherichia coli, σS has been reported in Pseudomonas species and Legionella pneumophila. (78) σB in Bacillus subtilis σB factor is expressed by the sigB gene. It was found by a large number of independent studies that σB is a stationary phase master regulator and experiences increase in intracellular levels in response to a variety of stresses. Thus, like σS of Escherichia coli, σB is a general stress response regulator. (109-112) Unlike σS however, σB has a redundant transcription factor, but even though a redundant factor exists, making a sigB null mutant rendered the organism sensitive to stresses such as ph alterations, heat shock, osmotic and oxidative stress. (78) Given that σB is a master regulator, it is obvious that it is under strict regulation. The main repressor of σB is RsbW protein which remains bound to σB and it is the release of the sigma factor from RsbW which allows the induction of general stress response related genes. RsbW itself is under the control of RsbV which controls the state of phosphorylation of RsbW. In normal cells, RsbV itself is phosphorylated at a conserved serine residue and it cannot interact with RsbW. So, RsbW is free to interact with σB and prevent its function. But, when a cell is under stress, RsbV is dephosphorylated by different enzymes, which allows RsbV to interact with RsbW and dephosphorylate it. This releases σB from RsbW and a stress response is generated. (110,113-115) Thus, the activation of a stress response relies on the dephosphorylation of RsbV. Two phosphatases have been detected for RsbV activation - RsbP, which is activated when the cell experiences energy stress, and RsbU, which is activated when the cell experiences environmental stress. (116-118) Thus, a partner switch mechanism where a dephosphorylated RsbV forces RsbW to switch partners is what governs the activation of σB. Many other organisms possess a σB homologue such as Listeria monocytogenes, Staphylococcus aureus and Mycobacterium tuberculosis. (Mycobacterium has a σF factor which performs in a manner similar to σB) (119-121) Antimicrobial stress response - Antimicrobial stress responses can be induced by intended or off-target effects of antimicrobials. The most general response of bacteria to antimicrobial stress is to generate persister cells. Persistence is a state of existence of bacteria where upon being exposed to an antimicrobial, only a specific subset of the population survives. What is important to note is that these persistent cells do not experience an increase in their minimum inhibitory concentration for that specific antimicrobial. Thus, persistence is a population level phenomenon. Regardless of what antimicrobial is used, persistent cells can recover when the antimicrobial is removed. (122,123) Oxidative stress response, rpoS mediated general stress response and Stringent response are the major stress responses that have been implicated in generation of persistence. (123) It has been known for some time now that antimicrobials can have off-target effects. A well known example is of β-lactam antibiotics which can cause production of reactive oxygen species via a reaction known as the Fenton reaction which goes 2+ 3+ . as follows - Fe + H O Fe + HO + OH2 2 The generation of ROS can lead to the oxidation of OxyR and SoxR which activates the oxidative stress response. Further, general stress responses such as RpoS mediated stress response can be activated due to oxidative stress as alluded to earlier in the review. (124) Activation of the stringent response by antimicrobials also leads to large scale changes in transcription in Bacteria. Changes such as reduction in replication rate of bacteria and reducing the elongation rate of DNA caused by the stringent response can serve as mechanisms of persistence. (125) Beyond persistence, Sturgeon et al went to show that the stringent response can induce acquisition of resistance in a population by promoting horizontal gene transfer. Using a PintI1 βgalactosidase reporter (where IntI1 codes for the integron integrase which allows acquisition or exchange of antibiotic resistance genes) they observed that 6 deletion of RelA gene caused a reduction in the levels of IntI1 expression in a biofilm, thus indicating that the stringent response contributes to the activation integron integrase and thus, can serve as a method of acquisition of antimicrobial resistance. (126) Another response of bacteria to antimicrobial stress can be a bet hedging strategy, termed ‘Hypermutation’. Hypermutation is a phenotype of bacteria where upon exposure to antibiotics, the inherent rate of acquisition of de novo mutations in bacteria increases in what is called ‘Stress induced mutagenesis’. Until a suitable adaptation is made, this increased rate of mutation persists, and when the adaptation is complete, normal rates of mutation are restored via anti-mutator mechanisms. An obvious mechanism of producing such mutations exists in the SOS response which depends on the activation of an error prone DinB polymerase which can induce such mutations. There also exist permanently hypermutable strains which contain mutator alleles which have mutations in genes responsible for making DNA repairs such as mutS and mutY both of which are involved in mutability repair of DNA. Such temporary hypermutators and permanent mutators have been isolated from clinically relevant pathogenic strains such as Streptococcus pneumoniae and Escherichia coli. (127,128) Thus, a wide variety of responses can be generated in response to antimicrobial based stresses, ranging from passive persistence to active mechanisms which can generate resistance. With this, the general stress responses of bacteria have been covered. It is time then to take a closer look at the molecular players which activate the effector enzymes and molecules that produce these responses. The molecular players: First, we shall learn about the major master regulators which cause widespread changes in bacterial metabolism during induction of stress, followed by the myriad of second messenger molecules which have been shown to cause large scale alterations themselves. In favour of the limited scope, the major effectors under these master regulators will be covered and only their large scale effects will be mentioned. The master regulators OxyR and SoxR The proteins under the OxyR and SoxR regulons do not overlap much even though both of them respond to oxidative stress. One of the proteins that is activated by both OxyR and SoxR is the fur gene which expresses a repressor of Fe3+ ion uptake, hence protecting the cell from OH• radicals. (127) Beyond that, the proteins under the two regulons vary, but most of them are antioxidants. Some examples of antioxidants under OxyR are katG (hydroperoxidase I), ahpCF (alkyl hydroperoxide reductase), gorA (glutathione reductase) and grxA (glutaredoxin). (68) OxyR also activates OxyS which is a small RNA which protects the cell against mutagenesis and also acts as a repressor for rpoS and fhlA. In contrast, SoxR activates a different set of genes. SodA (superoxide dismutase) represents the major part of the antioxidant arsenal of SoxR regulon. Other members of the regulon include nfo (DNA repair enzyme) and O2•resistant enzymes such as acnA product aconitase. (69,70,128) Besides, ROS, SoxR is also involved in protecting the bacterium against nitro compounds by expressing nitroreductase A. SoxR also activates efflux pumps via the activation of acrAB gene coded drug efflux pump and tolC encoded OMP. (129-131) Thus, both OxyR and SoxR induce a wide variety of genes. However, there are some genes which protect the bacterium against oxidative damage which are clearly not under the control of OxyR and SoxR. Some of these genes are katE which expresses hydroperoxidase II, recA, mutY and mutM which are involved in DNA damage control and the sodB and sodC genes which code for superoxide dismutases themselves. (132-135) There is also some overlap of the SoxR and OxyR regulons with the σS regulon. KatG and gorA (OxyR induced) and acnA (SoxR) are some of the genes which are activated by σS. σS has been shown to be an inducer of SodC. (135-137) σS and σBSince the responses in the general stress response are diverse, we shall tackle them section by section. σS response is directly involved in oxidative stress response as alluded to earlier in this review. The search for σS induced oxidative stress effectors began when it was discovered that rpoS null mutants were sensitive to hydrogen peroxide. (98) All the genes conferring oxidative stress resistance under the control of σS have already been covered in the OxyR and SoxR regulon section. A large part of the general response is spearheaded by the production of trehalose by the oprAB operon, which serves as a stress resistor against osmotic, desiccation and heat shock stresses. (138-140) The ecp-htrE operon also provides similar protections to the bacterium. (141) The σS response can also code for GABA antiporter by inducing gadC and gadB which allows a bacterium to tolerate altered pH. (142) Besides just protection against stress, the general stress response can also induce apoptosis of a bacterium in a population such that the nutrients from the dead cell can support the cells nearby. The ecnAB operon can cause such a response. (143) Pathogenic bacteria experience decreased virulence when the rpoS is repressed. Further, specific cases have been registered where σS is required for a pathogen to survive in the host or remain virulent. An example of that would be the esp genes which are required by enteropathogenic and enterohaemorrhagic Escherichia coli to express a type III toxin/antitoxin system. (91,144) Like σS, σB also penetrates various stress responses. It induces class II genes of the heat shock response and some class III genes such as clpP and clpC. Further, it controls genes such as KatB and KatX which are catalases that are expressed during starvation and oxidative stress response. Beyond that, σB also supports the osmotic stress response by inducing opuE gene and protects against desiccation by expressing GsiB. (145-147) An important function of σB response is causing drug efflux by activating the bmr operon genes 7 bmrU, bmr and bmrR which code for an efflux transporter that removes a diverse range of drugs such as Rifampin and Streptomycin which have been shown to induce σB response. (148-150) Second messengers Second messengers are molecules which transfer signals from a receptor to a target. These molecules diffuse rapidly in the cytoplasm of the bacterium and help in mounting a large scale response to various environmental cues. Here we shall cover the major second messenger molecules which are involved in stress based responses in bacteria. (p)ppGpp (p)ppGpp was discovered by Cashel and Gallant as a ‘magic spot’ on an autoradiogram of a starved Escherichia coli cell. They exposed Escherichia coli cells to radioactive phosphorus (P32) and compared acid soluble substrates separated via thin layer chromatography and saw that a spot was visible between GTP and the origin only for cells which were amino acid starved. This magic spot was later found to be Guanosine nucleotides pppGpp and ppGpp. (151) Since then, various studies have revealed that these molecules perform functions way beyond just the stringent response and apparently have their hand in almost all pockets of the bacterial system. The functions of (p)ppGpp can be divided into three parts (I) Reprogamming transcription - (p)ppGpp along with DksA can bind directly to RNA polymerase and can cause allosteric changes in the RNAP which affect the stability of the RNAP-promoter complex. (152,153) By causing changes in the RNAPpromoter complex stability, large scale alterations in transcription are achieved. Besides that, two independent studies by Corrigan et al and Zhang et al showed that (p)ppGpp can interact with various ribosome-associated GTPases such as Era in both gram positive and gram negative bacteria. (154,155) By doing so, (p)ppGpp can affect the assembly of ribosomes themselves as these GTPases are essential assembly factors in bacteria. (156) Beyond that, (p)ppGpp can control the cellular pools of GTP by inhibiting enzymes such as HprT and Gmk that contribute to GTP biosynthesis, which leads to decreased levels of GTP available in the cell. As a result, reduction in GTP levels can affect processes such as elongation of DNA replication. (157) This allows for the perfect segue to the next point. (II) Stalling DNA replication - (p)ppGpp molecules can stall DNA replication in two ways, directly by inhibiting initiation and indirectly by reducing the cellular levels of GTP and inhibiting elongation. (157) DnaG is the target enzyme for (p)ppGpp and by binding to it, the process of RNA primer synthesis which is necessary for DNA replication is inhibited. As alluded to earlier, the process of elongation is indirectly inhibited by decrease in cellular levels of GTP. The result is a dramatic decrease in the growth and multiplication of bacteria. Whats intriguing about this stalling is that conventionally, stalled replication forks recruit RecA for resolution, however, when replication is stalled by (p)ppGpp, no such recruitment occurs and thus DNA replication comes to a complete halt. (158) (III) Affecting various pathways - Beyond the above two processes, (p)ppGpp is a known contributor to bacterial persistence, tolerance and resistance. It was shown in Escherichia coli that a mutation in the hipA gene resulted in generation of persistence at a high rate. In an independent study, (p)ppGpp was found to be necessary for the generation of this persistence phenotype. (159,160) Studies with multiple bacteria has also implicated (p)ppGpp in generation of tolerance. Khakimova et al proved in Pseudomonas aeruginosa that (p)ppGpp is necessary for generation of tolerance to both antibiotics and oxidative stress. They generated ΔrelAΔspoT cells and observed that cellular catalases such as katA and katB underperformed. A related decrease in tolerance was observed when the bacterium was challenged with colistin and ofloxacin. (161) Resistance and (p)ppGpp go hand in hand as well. Earlier in this review, it was mentioned that the stringent response can induce horizontal gene transfer as a strategy to acquire a resistance conferring gene. Beyond that, some studies on Methicillin-resistant Staphylococcus aureus demonstrated that induction of (p)ppGpp can increase the resistance of this bacterium to βlactam antibiotics. (162,163) Besides the prototypical alarmones, recently pGpp and (p)ppApp have been discovered. pGpp was discovered in Bacillus anthracis and using a differential radial capillary action ligand assay, it was unearthed that pGpp binds to different targets as compared to (p)ppGpp an example of which is that pGpp interacts with effectors involved in purine biosynthesis. (164) In stark contrast, (p)ppApp was discovered when a type VI toxin-antitoxin was studied in Pseudomonas aeruginosa. The toxin Tas1 when invading other bacteria led to the overproduction of (p)ppApp which depleted the cellular pools of the target cells, leading to their death. (165) Cyclic dinucleotides c-di-GMP and c-di-AMP are two molecules which play extensive roles in bacterial stress responses. c-di-GMP -The proteins that interact with c-di-GMPs have c-di-GMP interacting domains. Some of the important proteins with these domains are Class I and Class II c-di-GMP riboswitches, proteins with a PilZ domain and some proteins which have either a GGDEF or an EAL domain. (166-168) c-di-GMP is responsible for shifting the lifestyle of the bacterium towards the formation of a biofilm by binding to YcgR. This YcgR-cdi-GMP complex then interacts with flagellar proteins FliG and FliM and inhibits the motility of a bacterium (which was assessed in the study by checking the motility of the bacterium on soft agar after removing a known repressor of c-di-GMP, YhjH). (169) Other effector functions of c-di-GMP include controlling development, an example of which is Caulobacter crescentus, a bacterium which has two life stages, a swarmer and a stalked stage. Some of the proteins involved in the transition of the bacterium from swarmer 8 to stalked stage are PleD and DgcB, both of which are diguanylate cyclases i.e. the enzymes which synthesise c-di-GMP. Another piece of evidence for c-di-GMP as an effector molecule is that the levels of c-di-GMP are lower in swarmers as compared to stalked cells. (170) This second messenger is also a major player in bacterial virulence. An important example of this is Clostridium difficile which depends on c-di-GMP to switch from a motile state to an adhesion state where it expresses multiple adhesins such that it can attach to the intestinal wall of the host. (171) On top of that, the second messenger is also involved in the regulation of toxin production by this pathogen. c-di-GMP was found to inhibit a gene called sigD, a gene which when ectopically expressed, increased the toxin production. (172) c-di-GMP was also found to be involved in protection against oxidative stress in Mycobacterium smegmatis in which it was directly responsible for the phosphorylation of devR operon which when deleted, made the bacterium sensitive to oxidative damage. (173) c-di-AMP - c-di-AMP on the other hand has only a few recognised interactions, even fewer that are relevant to virulence and survival. (174) DarR of Mycobacterium smegmatis is known to interact with c-di-AMP and is also involved in cold shock response. (175) A study showed that deletion of a gene (pdeA) in Streptococcus mutans increased the amount of c-di-AMP which promoted biofilm formation. (176) Much is still unknown about the functions of this second messenger so it is a ripe area for scientific exploration. With the molecular players covered, we shall now move on to the final section in which we look at how stress response allow bacteria to survive antimicrobial challenge. Bacteria Fighting back: In this part of the review, information regarding how stress responses manifest in the form antimicrobial persistence, tolerance and resistance will be covered. Again, given the limited scope, only mechanisms which are under the control of stress responses will be covered. Contribution of stress responses in development of antimicrobial persistent, tolerant and resistant bacterium Antimicrobial persistence, tolerance and resistance are all phenotypes which allow survival of bacteria when exposed to antimicrobials. A variety of mechanisms can lead to the development of these phenotypes. Bacteria which have not been exposed to an antimicrobial can already be in a persister or tolerant state by the induction of stress responses as part of a biofilm which can cause both nutritional and oxygen deprivation which can in turn lead to the activation of stringent response and oxygen starvation, both of which inhibit the active growth. This can make a bacterium persistent, tolerant or in some cases, even resistant to an antimicrobial. It was observed in Pseudomonas aeruginosa that cells which were largely deprived of oxygen via using oxygen electrodes. When these biofilms were exposed to antimicrobials such as tobramycin and ciprofloxacin, cells in early stage biofilms (4h) were almost completely wiped out as compared to that of comparatively elder biofilms (48 h) in which a much larger portion of cells survived. (177,178) Nutrient starvation can lead to the activation of stringent response and its effector, the (p)ppGpp alarmone, which also works to inhibit bacterial growth. Obviously then, in such a state a bacterium would become tolerant to antimicrobials as was seen in Escherichia coli which was shown to be tolerant to βlactam antibiotic exposure when the stringent response was active. (179,180) Further, (p)ppGpp renders penicillin binding protein 2 redundant and thus causes resistance to mecillinam. (181) Oxidative stress has been implicated as the go to method of killing by bactericidal antibiotics, many of which generate OH• radicals that cause large scale damage to the target bacterium. (182-184) It has been shown that deletion of genes which respond to oxidative stress can make a bacterium susceptible to antimicrobials. (185) Envelope stress has been registered as a culprit in this process as well. When this response is active, especially CpxAR, cells experience reduced susceptibility to a variety of antimicrobials such as β-lactams, aminoglycosides and even cationic antimicrobial peptides. (185,186) This largely occurs because of the activation of drug efflux pumps that reduce the intracellular concentration of antimicrobials by removing them actively. (185,187,188) In a comprehensive study on two component systems (TCS), it was proved that overexpression of 15 of the TCSs led to multi drug resistance in Escherichia coliI. Some examples of such TCSs are baeR, soxR, ompR and RcsB, all of which led to upregulation of drug efflux related genes and thus led to immediate increase in the MIC of the bacterium. (188) The general stress response regulated by σS and σB is in on the action as well. A mutation in the sigB gene led to Bacillus subtilis becoming susceptible to rifampin. (189) Its overexpression led to resistance to β-lactam antibiotics in Staphylococcus aureus. (190) σE was shown to restore some tolerance to ofloxacin in Pseudomonas aeruginosa when it was overexpressed in a ΔLasIR strain (suppresses quorum sensing and makes the bacterium highly sensitive to ofloxacin). (191) Even beyond all of these, stress induced mutagenesis (hypermutation) is a major source of development of antimicrobial resistant mutations which are heritable. This follows a set pattern where antimicrobial stress leads to DNA damage which activates SOS response, along with σS based general and σE based envelope stress response, all of which lead to the use of an error prone DNA polymerase for repair (DinB - SOS; DNA Pol II and IV - σS) which leads to a rapid and transient increase in mutation rate which increases the probability of achieving a mutation which can make the bacterium resistant to the antimicrobial responsible for the stress. (192-194) Hence, stress responses to a large degree contribute to the development of persistent, tolerant and resistant phenotypes across a large variety of bacteria. 9 Specific contribution of oxidative stress induced by antimicrobials to development of resistance It has been known for a while that bactericidal drugs can kill a bacterium in ways other than just the intended method. A study in 2007 by Kohanski et al argued that ROS mediated killing is a common mechanism of action in all bactericidal antimicrobials. (195) A mechanism for how ROS could induce cell death was known by that time. Imlay et al had already described in 1988 that hydroxyl radicals can kill a bacterium by causing oxidative damage, affecting Fe-S clusters and even disrupting the TCA cycle, all of which combined can overwhelm a bacterium and lead to its death. (196) Studies arguing Kohanski's hypothesis arrived quickly which grew bacteria in anoxic conditions and tested the lethality of antimicrobials and found that even in the absence of ROS, bacteria were still killed, which was the anti-thesis of Kohanski’s idea. (197) In any case, what is important that ROS can be generated by antimicrobials and can contribute to both bacterial death and development of resistance. Multiple studies made the argument that antimicrobials employ multiple mechanisms by which they cause cell death, of which, ROS generation is significant. (198,199) A recent article by Hong et al confirmed this significance by measuring cell death occurring in bacteria after the antibiotic stress was removed. To prove that it was ROS which was responsible for bacterial death, they supplemented the growth medium with bipyridyl which prevents the generation of ROS. The supplementation by bipyridyl led to a 30-fold increase in the survival rate of the bacterium as compared to the control, all in the absence of the original stressor, nalidixic acid. (200) Thus, ROS generation plays a role in bactericidal activity. Beyond just the oxidative damage, ROS can lead to DNA damage and can lead to double stranded DNA breaks. It is a major inducer of the SOS response, which itself employs RecBCD for repair of double stranded break repair which can be unsuccessful and hence lead to lethality. (201,202) How does all of this contribute to the development of antimicrobial resistance? Oxidative stress can activate oxidative stress response, the general stress response and SOS response as mentioned in the review already. These stress responses themselves in the previous section have been implicated in multiple mechanisms of generation of persistence, tolerance and resistance by using strategies such as drug efflux pumps and by inducing hypermutation via the use of error prone DNA polymerases. It is obvious then how oxidative stress response could be connected to generation of antimicrobial resistance. There is a glaring lack of studies which indicate this correlation however, so this still remains a hypothesis. It leaves a hole in the literature which needs to be plugged. The epigenetic perspective on generation of antimicrobial resistance - were too high to just occur via spontaneous mutations, bolstered by the information that said resistance can revert and the bacterium can become sensitive, which is the tell tale sign of an epigenetic alteration. It was shown in a study by Adam et al that low level exposure to antibiotics led to change in the expression of multiple genes which led to increase in antibiotic resistance and that the survival rate increase by about 5 fold when Dam methylase, an enzyme which methylates the adenine of GATC, was upregulated. (203) This phenomenon was named adaptive resistance. Since then, some studies have tried to look at the epigenetic basis of acquisition of antimicrobial resistance. A study by Cohen et al showed that removal of Dam methylase from Escherichia coli made it sensitive to the action of β-lactam antibiotics. (204) There is a lack of studies which explore the impact of epigenetics and its contribution to rapid development of resistance, and thus, there lies another area where research could be conducted. Conclusion and Future perspectives: The aim of this review was to understand how a bacterium survives in a world with so many stresses and fluctuations. Section by section, we explored the canonical stress responses, followed by the master regulators and effectors employed in the generation of said responses followed by an obstacle which will become more daunting in modern times, the problem of bacterial persistence, tolerance and resistance. We have achieved some level of understanding of how bacteria respond to stress and how those stress responses allow bacteria to escape antimicrobials. Having said that, there are still a lot of questions which remain unanswered and thus, a lot of work remains to be done. Questions ranging from how (p)ppGpp synthesis works in its entirety to how oxidative stress can contribute to the generation of antimicrobial resistance remain unanswered. Beyond that, an entire field of epigenetic changes in bacteria remains somewhat unexplored even though there is some evidence which suggests that epigenetics might be involved in the rapid escape of bacteria from the wrath of antimicrobials. A huge question which the literature shows no instance of addressing is, how exactly is the presence of antimicrobials sensed? What characteristic of antimicrobials awakens the bacterium which then begins to retaliate? What are the molecules involved in such a sensory process? Answering these questions could revolutionise the field of drug development as it could provide us with a roadmap of what pitfalls to avoid when making a drug and thus kill bacteria while they are none the wiser. With all said and done, it is imperative to reiterate the fact that research in acquisition of antimicrobial resistance is of extreme importance. With antimicrobial resistant pathogen related deaths trending upwards, a hypothetical clock is ticking and we must endeavour to have all the answers and a plan of action ready before the alarm rings. Epigenetic inheritance was birthed as an idea when it was observed that the rates of acquisition of resistance 10 Lab report: Background: In Caulobacter crescentus, licensing of flagellation occurs via a regulator called MadA (motility and division regulator) which binds directly to FlhA in the flagellar basal body and releases a protein called FliX which binds to FlbD. This FliX-FlbD interaction allows the hierarchical process of flagellar biogenesis to move from class-II components to class-III components. (See schematic below) This is supported by the fact that in ΔmadA cells, the levels of class-II components (such as FliF and FliX) increases whereas the levels of class-III components (such as FlgE and FlgI) decreases. It is believed that the tethering of FliX to FlhA is what keeps it inactive and that its subsequent removal from FlhA due to the binding of MadA to FlhA introduces a conformational change in FliX which activates it and allows it to activate FlbD and thus, the genes downstream. Until now however, the conformational change which leads to the activation of FliX, hence leading to its binding to FlbD remains unknown. (205) The aim of the project is to elucidate this conformational change that occurs in FliX which leads to its activation. Regulation of Flix-FlbD activity by MadA during flagellar assembly. Reprinted from ‘An organelle-tethering mechanism couples flagellation to cell division in bacteria’ by Siwach et al, Developmental Cell, 2021 Objectives of the project: 1. To develop a mutant FliX which can activate FlbD without the intervention of MadA. 2. To isolate and crystallise the FliX mutant and study its structure. Developing a FliX mutant: A WT FliX gene was ligated to pmT335 high copy number plasmid which has a Gentamicin resistance marker. The plasmid is passaged through Escherichia coli XL red cells which are ΔmutHLS (error prone replication due to impaired mismatch repair.) By doing so, random mutations shall be introduced in the plasmid due to the error prone DNA replication. 11 These plasmids harbouring random mutations would be transformed into ΔmadA and ΔmadAΔfliX Caulobacter crescentus cells. The cells carrying these mutant plasmids shall then be screened for motility by performing a soft agar motility assay and looking for colonies which show the development of swarmer cells. 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