Another Cell Bites the Dust

by Janie
 

Almost a century ago, Walther Goebl and Oswald Avery observed that Streptococcus pneumoniae cells lysed upon entering stationary phase, but the mechanism behind this autolysis remained elusive.
 

Figure 1. Transmission electron micro­scopy image of a lysing bacterium. Source

This challenge is precisely what a study by a team of researchers at Harvard Medical School, the University of Sheffield, and the Howard Hughes Medical Institute tackled. The study took a closer look at the role of peptidoglycan (PG) hydrolases in bacteriolysis. PG hydrolases, also known as autolysins, cleave bonds in peptidoglycan networks, providing bacteria with a means of cell wall remodeling required for growth and division. Their double-edged role in poking holes in the cell wall was of interest: PG hydrolases and their wall-cleaving activity could be essential to normal growth under one set of conditions, but lethal under another. Piecing together the mechanism of PG-hydrolase-mediated lysis is of particular interest, as PG hydrolases are also implicated in beta-lactam-induced lysis. Understanding exactly how antibiotics like penicillin subvert bacterial defenses, undermine the integrity of the cell wall, and ultimately cause lysis of pathogens would be valuable in advancing therapeutic developments.
 

Introducing LytA and TacL: an enzyme that wears two hats, and its inhibitor

The researchers began to unravel the role of PG hydrolases in autolysis. S. pneumoniae was the perfect microbe for the job, as the bacterium conveniently contains only one PG hydrolase, LytA, and is prone to autolysis after extended time in stationary phase.
 

Because LytA levels stay constant throughout growth, from exponential phase to stationary phase to autolysis, the team hypothesized that there must be an additional regulatory component that inhibits LytA during exponential growth. Such a regulator would be required for growth in cells with functional LytA but unnecessary in cells lacking LytA. They performed a Tn-seq screen for regulators of the PG hydrolase and found that the gene tacL was required for survival in wild-type S. pneumoniae but dispensable in ΔlytA strains. They confirmed these results by comparing growth curves for strains with tacL on and off, finding that cells lacking TacL underwent premature autolysis during exponential phase. Thus, TacL must exert inhibitory effects on LytA during exponential growth.
 

Figure 2.  Diagram of Streptococcus pneu­moniae cell envelope. The cell wall pepti­do­glycan (PG) (blue) contains Wall Teichoic Acid (WTA) polymers and the lipid bilayer contains Lipoteichoic Acid (LTA). The con­stituents of the repeating unit in LTAs and WTAs are indicated; Cho, choline; GlcNac, N-acetylglucosamine; Rbo, ribitol; ATTGal, 2-acetamido-4-amino-2,4,6-trideoxygalactose; Glc, glucose; Phos, phos­phate. . Source

The next step was to figure out how exactly TacL reins in LytA’s cell-wall-cleaving activity. One possibility was that TacL could impede secretion of LytA during exponential growth. If this were true, then cells with and without TacL should both be equally sensitive to the exogenous addition of purified LytA. In alignment with the team’s prediction, the addition of LytA to ΔlytA cultures and to ΔlytA ΔtacL cultures in exponential growth resulted in rapid lysis of the latter cells. This showed that TacL protects cells from death-by-LytA at a point after LytA has already been exported to the cell surface.
 

Tipping the LTA-WTA scale

With two key enzymes in the books, the next step was to determine their relationship with potential cell envelope substrates. Lipoteichoic acids (LTAs), structural components of the lipid bilayer, and wall teichoic acids (WTAs), structural components of the cell wall, are suspected to be synthesized from the same undecaprenyl phosphate precursor in S. pneumoniae. A previous study found evidence that the membrane protein TacL attaches the teichoic acid bit of the precursor molecule to a glycolipid anchor, producing LTA (hence, TacL standing for techoic acid ligase).
 

The team first confirmed that TacL is required for LTA formation. They measured the levels of LTA and WTA in the following strains during exponential growth: WT, ΔlytA, ΔlytAΔtacL, and a double mutant with a Zn-inducible tacL allele, ΔlytAΔtacL+tacL. The results? When LTA levels were down, WTA levels were up, and vice versa. The ΔlytAΔtacL cells entirely lacking a source of TacL produced no LTA, but the amount of WTA detected in these same cells was significantly higher than that of any of the other three strains. This suggested that WTA and LTA synthesis are inversely related, additional evidence that they compete for the same undecaprenyl phosphate precursor.
 

So far, the team had elucidated that the absence of TacL during exponential growth causes the switch from LTA to WTA synthesis and triggers an autolysis pathway involving LytA. Because LytA has domains for cell wall cleaving amidase activity and for binding with teichoic acids, they guessed that LytA might localize to the cell wall in the absence of TacL, granting LytA more ready access to its WTA substrate to cause cell wall degradation and subsequent cell lysis.
 

This hypothesis was put to the test. The team tracked LytA localization patterns during three stages of growth—exponential growth, stationary growth, and late stationary phase when autolysis occurs—in both TacL+ and TacL- strains (in an inactivated LytA background, LytA(H26A), to avoid complications arising from autolysis). As anticipated, the TacL+ strain had more LTAs than WTAs during exponential growth, and the majority of the LytA(H26A) were still found in the membrane. But as the cells reached late stationary phase, LTA levels dropped while WTA levels soared, and LytA(H26A) was now localized to the cell wall, where it would be busy chewing away at the matrix for impending lysis.
 

It seemed likely that penicillin-induced autolysis would follow a similar pathway. Indeed, repeating the experiment with LytA(H26A) cells either exposed or not exposed to penicillin confirmed this guess, demonstrating the same switch in LTA to WTA synthesis and localization of LytA to the cell wall.
 

Figure 3. Transposon insertions in tacL were found much more frequently in cells lacking lytA. Source

Another enzyme behind the curtain

The rapid disappearance of LTAs observed in the last experiment was an object of intrigue: perhaps there was another actor in play, removing existing LTAs from the cell membrane. So, the team monitored the LTA levels of LytA^- cells in the membrane versus supernatant and compared with that of LytA⁺ cells. At the start, LTAs were detected in the membrane. However, upon entry into late stationary phase or exposure to penicillin, LTAs were hardly detectable in the membrane. There was also more choline-containing material recovered from the supernatant, likely sourced from the LTAs. This suggested that LTAs are released from lysing cells, allowing LytA to direct its energies toward associating with WTAs to cleave the cell wall.
 
 

Figure 4. Model for LytA regulation. Dur­ing exponential growth, TacL-dependent LTA synthesis dominates over LCP-dependent production of WTA. LytA bound to the phosphocholine moeities (purple/black balls) on LTA is sequestered away from WTA preventing LytA from targeting the cell wall. Upon entry into stationary phase or exposure to cell wall targeting anti­biotics (penicillin), TacL is degraded in an FtsH-dependent manner leading to a red­uction in LTA synthesis and an increase in WTA levels. Association of LytA with newly synthesized WTA leads to cell wall cleavage and lysis. Source

The next part of the puzzle was to uncover how the LTA to WTA synthesis switch is initiated. Suspecting that TacL was the primary regulatory target, the team monitored the effect of tacL overexpression on autolysis induction. In both late-stationary-phase cultures and penicillin-treated cultures, tacL overexpression was found to prevent autolysis, which further suggested that a reduction in TacL levels does indeed cause the autolytic switch from LTA to WTA synthesis.
 

Upon FLAG-tagging  TacL to track its levels in different stages of growth with and without penicillin exposure, the team found that TacL levels decreased with the start of autolysis only in the strain with functional LytA. The half-life of TacL-FLAG in ΔlytA dropped upon entering late stationary phase and exposure to penicillin, suggesting that TacL is degraded during autolysis. So, what protease is responsible for getting rid of TacL? A second Tn-seq screen pinpointed the gene ftsH. FtsH is an ATP-dependent zinc metallopeptidase that has already been shown to degrade membrane proteins in a different study, so it was reasonable to suspect that FtsH could be the TacL-degrading culprit.

Confirming their hunch, a comparison of levels of TacL-FLAG, LTA, and WTA in FtsH⁺ and FtsH^- cells demonstrated that FtsH^- cells displayed none of the usual signs of impending autolysis: no decrease in TacL-FLAG levels, no switch from LTA to WTA synthesis: zilch, unlike the FtsH⁺ cells.
 

The pathway resolved

So, LytA degrades the cell wall, TacL is the ligase that synthesizes LTAs, and FtsH degrades TacL. At last, the model for S. pneumoniae lysis comes together. In autolytic conditions, FtsH’s degradation of TacL decreases the levels of LTAs present (which is paired with an increase in WTAs). Without LTAs to bind and sequester LytA, LytA is now able to localize to the cell wall, where it can then associate with WTAs to ignite its cell-wall-cleaving activity. In this way, the cell wall is dismantled.
 

The next step is to zero in on what triggers FtsH-mediated degradation of TacL. This new understanding could prove to be a trump card in the perpetual quest to bring down the bacterial cell wall of pathogens. 

 

Reference

Flores-Kim, J., Dobihal, G. S., Fenton, A., Rudner, D. Z., & Bernhardt, T. G. (2019). A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis. eLife, 8, e44912. doi:10.7554/eLife.44912