Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide

Abstract

To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

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

“…Next, Mtb-associated LysoTracker intensity and Mtb intrabacterial pH were analysed by automated high-content microscopy ( Figure S1 ). A quantitative analysis in Mtb-infected MDM (median CTRL = 422.6; IQR CTRL = 334.9 and median ConA = 241.6; IQR ConA = 64.5, respectively) and Mtb-infected iPSDM (median CTRL = 1964.2, IQR CTRL = 1006.0 and median ConA = 929.3; IQR ConA = 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 (Δmedian pH-GFP = 0.035 and 0.016 respectively), suggesting that intracellular acidification does not impact Mtb intrabacterial pH in human macrophages.…”

“…Strikingly, from the four different antibiotics tested, PZA was the only one able to induce changes in Mtb pH-GFP ratio, providing evidence that PZA displays intrabacterial pH-disruptive activity in Mtb-infected human macrophages (mean normalised pH-GFP ratio of 0.155; p -value ≤0.001) ( Figure 3C ). 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 host-microenvironments ( 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). Mtb can transit through neutral and acidic environments multiple times during the infection cycle (Bernard et al, 2020; Schnettger et al, 2017), however it remains unclear how these spatial and temporal changes affect the intracellular activity of PZA and its impacts on intrabacterial pH homeostasis.…”

The Transporter-Mediated Cellular Uptake and Efflux of Pharmaceutical Drugs and Biotechnology Products: How and Why Phospholipid Bilayer Transport Is Negligible in Real Biomembranes

“…This can involve both the targets within the microbe and the host’s transporters when (as is common, e.g., [ 468 , 469 , 470 , 471 , 472 , 473 , 474 , 475 , 476 , 477 , 478 , 479 , 480 , 481 , 482 , 483 , 484 , 485 ]) the infective agents reside intracellularly. A particularly clear example is given by Mycobacterium tuberculosis , the causative agent of TB, where the very striking lack of correlation between in vivo and in vitro drug potencies is easily and necessarily explained via transporter activities [ 139 , 151 , 486 , 487 , 488 ]. Many orally prescribed antibiotics enter the host via SLC15 family members [ 489 , 490 ], while some of the relatively few known microbial uptake transporters for anti-infectives are listed in Table 1 .…”