Bacterial growth and morphogenesis are intimately coupled to expansion of peptidoglycan (PG), an extensively cross-linked macromolecule that forms a protective mesh-like sacculus around the cytoplasmic membrane. Growth of the PG sacculus is a dynamic event requiring the concerted action of hydrolases that cleave the cross-links for insertion of new material and synthases that catalyze cross-link formation; however, the factors that regulate PG expansion during bacterial growth are poorly understood. Here, we show that the PG hydrolase MepS (formerly Spr), which is specific to cleavage of cross-links during PG expansion in Escherichia coli, is modulated by proteolysis. Using combined genetic, molecular, and biochemical approaches, we demonstrate that MepS is rapidly degraded by a proteolytic system comprising an outer membrane lipoprotein of unknown function, NlpI, and a periplasmic protease, Prc (or Tsp). In summary, our results indicate that the NlpI-Prc system contributes to growth and enlargement of the PG sacculus by modulating the cellular levels of the cross-link-cleaving hydrolase MepS. Overall, this study signifies the importance of PG cross-link cleavage and its regulation in bacterial cell wall biogenesis.bacterial morphogenesis | peptidoglycan | regulated proteolysis | MepS | NlpI-Prc P eptidoglycan (PG or murein) is a unique and essential constituent of eubacterial cell walls, thus making it an excellent target for several antimicrobial agents. It is a single, large, extensively cross-linked macromolecule that forms a mesh-like sacculus protecting cells against intracellular turgor pressure in addition to conferring cell shape. Structurally, the PG sacculus is made up of linear glycan strands cross-linked to each other by short peptide chains forming a continuous layer around the cytoplasmic membrane. The glycan strands are made up of alternating N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG) disaccharide units in which NAM is covalently attached to a peptide chain containing 2-to 5-amino acid residues, with the pentapeptide consisting of L-alanine (ala)−D-glutamic acid (glu)−meso-diaminopimelic acid (mDAP)−D-ala−D-ala. Normally, D-ala of one peptide chain is cross-linked to mDAP of another peptide chain of an adjacent glycan strand, resulting in an extensively cross-linked single-or multilayered sacculus (1).Because the murein sacculus totally encircles the cytoplasmic membrane, growth of a cell is tightly coupled to expansion of PG. Growth of the PG sacculus is a dynamic and coordinated event requiring concerted action both of murein hydrolases that facilitate cleavage of cross-links for the insertion of nascent murein material and of synthases that catalyze cross-link formation between adjacent glycan strands (Fig. 1) (2, 3).Escherichia coli encodes multiple PG synthases that catalyze the formation of D-ala−mDAP cross-links in the PG sacculus. The class I enzymes (PBP1a and PBP1b, encoded by mrcA and mrcB, respectively) are bifunctional and possess both glycosyl transferase (GT) and tr...

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin–based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti–programmed cell death-1 (anti–PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin–based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.

Following infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), most human hosts are able to contain the infection and avoid progression to active TB disease through expression of a balanced, homeostatic immune response. Proinflammatory mechanisms aiming to kill, slow and sequester the pathogen are key to a successful host response. However, an excessive or inappropriate pro-inflammatory response may lead to granuloma enlargement and tissue damage, which may prolong the TB treatment duration and permanently diminish the lung function of TB survivors. The host also expresses certain anti-inflammatory mediators which may play either beneficial or detrimental roles depending on the timing of their deployment. The balance between the timing and expression levels of pro- and anti-inflammatory responses plays an important role in the fate of infection. Interestingly, M. tuberculosis appears to manipulate both sides of the human immune response to remodel the host environment for its own benefit. Consequently, therapies which modulate either end of this spectrum of immune responses at the appropriate time may have the potential to improve the treatment of TB or to reduce the formation of permanent lung damage after microbiological cure. Here, we highlight host-directed TB therapies targeting pro- or anti-inflammatory processes that have been evaluated in pre-clinical models. The repurposing of already available drugs known to modulate these responses may improve the future of TB therapy.