Intracellular localisation ofMycobacterium tuberculosisaffects antibiotic efficacy

Abstract

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of Mycobacterium tuberculosis (Mtb). However, how host cell environments affect antibiotic accumulation and efficacy remains elusive. Pyrazinamide (PZA) is a key antibiotic against TB, yet its behaviour is not fully understood. Here, by using correlative light, electron, and ion microscopy to image PZA at the subcellular level, we investigated how human macrophage environments affect PZA activity. We discovered that PZA accumulates heterogeneously between individual bacteria in multiple host cell environments. Crucially, Mtb phagosomal localisation and acidification increase PZA accumulation and efficacy. By imaging two antibiotics commonly used in combined TB therapy, we showed that bedaquiline (BDQ) significantly enhances PZA accumulation by a host cell mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy; explaining the potent in vivo efficacy compared to its modest in vitro activity and the critical contribution to TB combination chemotherapy.

Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis

“…The determination of PZA half maximal effective concentration (EC50) using a four-parameter logistic non-linear regression model showed that ConA co-treatment increased, by approximately 3.5 times, the amount of antibiotic required to efficiently inhibit 50% of Mtb growth in cellulo (49.5 ± 19.2 mg/L and 173.1 ± 35.2 mg/L, respectively) (Figure 4A-4B). These results agree with our previous observations showing that the use of v-ATPase inhibitors is able to counteract PZA/POA-mediated growth inhibition by impairing POA accumulation within the bacteria (Santucci et al, 2021). These experiments were also performed in another human macrophage model using iPSDM (Figure S6).…”

“…Next, Mtb-associated LysoTracker intensity and Mtb intrabacterial pH were analysed by automated high-content microscopy (Figure S1). A quantitative analysis in IQRCTRL = 334.9 and medianConA = 241.6;IQRConA = 64.5,respectively) and Mtb-infected iPSDM (medianCTRL = 1964.2, IQRCTRL = 1006.0 andmedianConA = 929.3;IQRConA = 1009.4, respectively) showed that the median Mtb-associated LysoTracker intensity was reduced by approximately 2-fold upon ConA treatment (Figure 1A and Figure 1C), confirming that endolysosomal acidification was impaired (Santucci et al, 2021). On the other hand, a quantitative analysis of Mtb intrabacterial pH in control or ConA-treated conditions were similar in both infected MDM (Figure 1B) and infected iPSDM (Figure 1D) with absolute median differences that were almost null (ΔmedianpH-GFP = 0.035 and 0.016 respectively), suggesting that intracellular acidification does not impact Mtb intrabacterial pH in human macrophages.…”

“…PZA/POA molecules require endolysosomal acidification to accumulate inside Mtb and display antimicrobial efficacy (Santucci et al, 2021). We hypothesized that this process is likely resulting from the conversion of POA (-) into its protonated form HPOA within acidic hostmicroenvironments (Figure 3D).…”

“…Ideally, anti-TB chemotherapy must include antibiotics with pharmacokinetic properties that allow agents to (i) penetrate lung tissue and infected cells, (ii) reach the wide-ranging subcellular compartments in which Mtb resides and (iii) be active in these specific microenvironments to finally display optimal antibacterial efficacy. Understanding how subcellular environments affect bacterial fitness (Huang et al, 2019;Rohde et al, 2007) but more importantly antibiotic localisation, exposure, and consequently efficacy against intracellular pathogens is crucial and have only recently begun to be investigated (Liu et al, 2016;Santucci et al, 2021).…”

“…By using correlative light electron ion microscopy (CLEIM) approaches we showed that PZA/POA molecules require phagosomal residency and acidification to efficiently accumulate within Mtb and display optimal activity within human macrophages (Santucci et al, 2021).…”