Amphipathic beta(2,2)-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae

We demonstrate in the current work that small cationic antimicrobial beta(2,2)-amino acid derivatives (Mw <500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (<5 mu M, i.e. <3.4 mu g/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the beta(2,2)-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these beta(2,2)-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic beta(2,2)-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-totreat intracellular pathogen.Peer reviewe

Treatment of <i>C</i>. <i>pneumoniae</i> elementary bodies with derivatives A1—A6.

<p>EBs were incubated with derivatives <b>A1</b>—<b>A6</b> at concentrations of 5 μM for 1 h. Suppression of infectivity of the EBs was determined after inoculation of HL cells and 72 h of incubation. (Results display the mean ± SEM).</p

Impact of delayed A1 and A2 administration and inhibition of infectious progeny production by the derivatives.

<p>(A) Inhibition of <i>C</i>. <i>pneumoniae</i> infectivity after delayed administration of 3 μM of derivatives <b>A1</b> and <b>A2.</b> (B) Inhibition of infectious progeny production by <b>A1</b> and <b>A2</b> at a concentration of 5 μM. (Results display the mean ± SEM; asterisk indicate significant difference, p < 0.05).</p

Schematic drawing of HL cell infection by <i>C</i>. <i>pneumoniae</i> and potential intervention points of β^2,2-amino acid derivatives.

<p>The cytosol is surrounded by the inner (grey) and outer (black) leaflet of the cell membrane. The possible sites of inhibition of β^2,2-amino acid derivatives during the <i>C</i>. <i>pneumoniae</i> infection cycle are indicated by lightning symbols. Inhibition of infection progress is indicated by double strikethrough.</p

Structures of the investigated β^2,2-amino acid derivatives A1—A6.

<p>All derivatives were isolated as di-trifluoroacetate salts.</p

Impact of β^2,2-amino acid derivatives on <i>C</i>. <i>pneumoniae</i> inclusion counts and host cell viability.

<p>(A) Screening of the β^2,2-amino acid derivatives <b>A1</b>—<b>A6</b> for anti-<i>C</i>. <i>pneumoniae</i> activity and cytotoxicity at concentrations of 5 μM. (B) Dose-response relationship of <b>A1</b> and <b>A2</b> for decreasing <i>C</i>. <i>pneumoniae</i> inclusion counts. (Results display the mean ± SEM).</p