ppe51 variants promote non-replicating Mycobacterium tuberculosis to grow at acidic pH by selectively promoting glycerol uptake

In defined media supplemented with single carbon sources, Mycobacterium tuberculosis (Mtb) exhibits carbon source specific growth restriction. When supplied glycerol as the sole carbon source at pH 5.7, Mtb establishes a metabolically active state of nonreplicating persistence known as acid growth arrest. We hypothesized that acidic growth arrest on glycerol is not a metabolic restriction, but rather an adaptive response. To test this hypothesis, we conducted forward genetic screens that identified several Mtb mutants that could grow under these restrictive conditions. All of the mutants were mapped to the ppe51 gene and resulted in three amino acid substitution – S211R, E215K, and A228D. Expression of the PPE51 variants in Mtb promoted growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, enhanced acid growth (eag) phenotype. Testing growth on other single carbon sources showed the PPE51 variants specifically enhanced growth on glycerol, suggesting ppe51 plays a role in glycerol uptake. Using radiolabeled glycerol, enhanced glycerol uptake was observed in Mtb expressing the PPE51 (S211R) variant, with glycerol overaccumulation in triacylglycerol. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication and reduced in virulence in activated macrophages as compared to resting macrophages. Recombinant PPE51 protein exhibited differential thermostability in the WT or S211R variants in the presence of glycerol, supporting the eag substitutions alter PPE51-glycerol interactions. Together, these findings support that PPE51 variants selectively promote glycerol uptake and that slowed growth at acidic pH is an important adaptive mechanism required for macrophage pathogenesis.


Introduction 46
During infection, Mycobacterium tuberculosis (Mtb) senses and adapts to a variety of immune cues including hypoxia 1,2 , nutrient starvation 3,4 , pH changes 5 , and nitrosative and 48 oxidative stress 6 . Exposure to these stresses can promote Mtb to establish slowed growth or a non-49 replicating persistent (NRP) state. NRP bacteria are tolerant to immune and antibiotic-mediated 50 killing 7-9 , therefore understanding mechanisms underlying NRP may promote new methods to 51 shorten the course of TB therapy. 52 Following macrophage infection, Mtb senses the mildly acidic pH of the phagosome and 53 broadly remodels its gene expression 10 . Adaptations to acidic pH include the induction of the 54 PhoPR regulon, induction of ESX-1 secretion, and remodeling of central metabolism and cell 55 envelope lipids 11 . Defects in adaptations to acidic pH reduce Mtb virulence in macrophages and 56 animals 12-15 , therefore, pH dependent adaptations are required for Mtb virulence. 57 Previous studies conducted by our lab sought to understand the interplay of acidic pH and 58 Mtb central metabolism. We observed that Mtb exhibits selectivity of the carbon sources on which 59 it can growth at pH 5.7 relative to pH 7.0. For example, Mtb incubated at acidic pH with glycerol 60 as a sole carbon source is restricted for growth and establishes a viable, metabolically active state 61 of NRP called acid growth arrest [16][17][18] . Acid growth arrest is observed on a variety of other carbon 62 tested this hypothesis using a CDC1551 transposon mutant library containing >100,000 was plated 276 on MMAT defined minimal media agar with glycerol at pH 5.7 and resulted in 98 transposon (Tn) 277 mutants and two spontaneous WT mutants 16 . These mutants were isolated and confirmed as 278 enhanced acid growth (eag) mutants based on their ability to grow well compared to native WT 279 Mtb at pH 5.7 in liquid MMAT supplemented with glycerol 16 . Interestingly, complementation 280 attempts with the Tn mutants did not restore growth arrest, and whole genome sequencing 281 identified spontaneous mutations in ppe51 in both Tn and WT mutant backgrounds 16 . To repeat a 282 saturating screen, in the absence of transposon mutagenesis, a second forward genetic screen was 283 performed on MMAT agar buffered to pH 5.7 with glycerol, using a larger bacterial population 284 (4x10 9 bacteria) in the Erdman Mtb strain ( Figure 1A). From the WT Erdman screen, 98 285 spontaneous eag mutants were isolated of which 52 were colony-purified and confirmed for 286 enhanced growth under acidic conditions in liquid MMAT containing glycerol ( Figure 1B, Figure  287 S1A-D). The eag isolates exhibited an up to ~4-fold increase in growth compared to WT Erdman 288 which exhibited complete growth arrest ( Figure 1B). Of these eag mutants, 24 were selected for 289 whole genome sequencing. Remarkably, all 24 isolates had single nucleotide polymorphisms 290 (SNPs) mapping to the ppe51 gene (Table 1). All mutations were non-synonymous (S211R, 291 A228D, and E215K) and were centrally located within a 50 bp region on the ppe51 gene ( Figure  292 S2). The S211R and A228D variants were also identified in the prior Tn mutant CDC1551 screen, 293 with E215K being a novel mutation found in the new Erdman screen. 294 295 ppe51 mutations are sufficient to overcome growth arrest. Given that ppe51 variants exhibit 296 enhanced growth at acidic pH, we investigated the function of the variant alleles in the presence 297 of the WT ppe51 allele. Overexpression constructs of WT or mutant ppe51 were transformed into 298 WT CDC1551 or WT Erdman Mtb strains carrying the native ppe51 allele. Overexpression strains 299 were grown in MMAT at pH 5.7 with glycerol as a carbon source. Overexpression of ppe51-S211R 300 and ppe51-A228D variants in WT Mtb resulted in significantly enhanced growth under acidic 301 conditions ( Figure 2A). In contrast, overexpression of WT ppe51 and the empty vector exhibited 302 complete growth arrest at pH 5.7. The E215K allele was also not sufficient at overcoming growth 303 arrest which may explain why it has only been observed once across two independent forward 304 genetic screens. Additionally, all overexpression strains grew equally well at pH 7.0 ( Figure S3), 305 showing that the observed growth phenotype is pH-specific. Interestingly, although the growth 306 phenotype with A228D overexpression resulted in enhanced growth at acidic pH, it grew at a 307 slower rate compared to S211R overexpression strains in both CDC1551 and Erdman 308 backgrounds. We examined individual variant alleles from the screen ( Figure 1B) and observed 309 that S211R variants significantly grouped together at a higher rate of growth compared to A228D 310 and E215K ( Figure 2B and 2C). Together, these results demonstrate that the eag mutations confer 311 a dominant, gain-of-function growth phenotype, and specific mutations are associated with 312 differential strength of the phenotype. 313 314 PPE51 variants selectively promote growth on glycerol. Based on the enhanced growth 315 phenotype that the variants exhibit at acidic pH, we hypothesized that this phenotype may be due 316 to ppe51 variants modulating mycomembrane permeability, resulting in enhanced nutrient uptake. 317 To test this hypothesis, we conducted an Ethidium Bromide (EtBr) assay looking at permeability 318 with WT Mtb overexpression constructs (empty vector, WT, and S211R) in both CDC1551 and 319 Erdman backgrounds. With the EtBr assay we did not observe differences in the rate of uptake 320 between the WT and eag overexpression strains in either CDC1551 or Erdman ( Figure S4), 321 suggesting that the growth phenotype is not due to a general increase in permeability. We then 322 hypothesized that the growth phenotype may be due to nutrient-specific uptake. We explored this 323 possibility by growing CDC1551 and Erdman overexpression strains (empty vector, ppe51 and 324 ppe51-S211R) in liquid minimal media (pH 5.7) in the presence of various growth-permissive (e.g. 325 pyruvate, acetate, cholesterol) and non-permissive (e.g. glucose, glycerol, propionate) carbon 326 sources 18 . After 20 days we found that enhanced acid growth was only observed with ppe51-S211R 327 in the presence of 10 mM glycerol, a normally non-permissive carbon source at pH 5.7, in both 328 CDC1551 and Erdman ( Figure 2D, Figure S5, Figure S6A-B). As expected, all overexpression 329 strains exhibited enhanced growth on permissive carbon sources in both Mtb backgrounds. 330 Notably, the ppe51 variants specifically promote growth on glycerol, but not glucose, another 331 proposed nutrient associated with PPE51-dependent uptake 28,41 , demonstrating the variants are 332 selective for glycerol. 333 334 ppe51 is not required for survival during acid growth arrest. Transcriptional profiling studies 335 previously conducted show that ppe51 is significantly induced during acid growth arrest 18 .We 336 hypothesized that ppe51 may be required for Mtb to promote survival when exposed to acid growth 337 arresting conditions. To test this hypothesis, a ∆ppe51 knockout strain was generated in both 338 CDC1551 and Erdman Mtb using the mycobacteria-specific ORBIT system ( Figure S7A) 48 . 339 Successful knockout of ppe51 was confirmed by sequencing the oriE and HygC junction sites of 340 ∆ppe51, PCR amplification of the entire knockout region, and RT-PCR ( Figure S7B-D). 341 Complementation constructs containing the native ppe51 promoter were introduced into ∆ppe51 342 carrying WT ppe51, variant ppe51 (S211R, A228D, E215K) and a double variant 343 (S211R+A228D). An empty complementation vector was also introduced into ∆ppe51. The presence of these constructs was also confirmed via RT-PCR ( Figure S7E). Growth curves of the 345 complementation constructs grown in growth arresting conditions showed that WT and empty 346 vector strains exhibit growth arrest at pH 5.7, whereas the variant complemented strains exhibited 347 enhanced growth in both CDC and Erdman ( Figure 3A, Figure S8A). Additionally, the S211R, 348 A228D, and S211R+A228D variant complemented strains grow slightly better compared to 349 E215K in both CDC1551 and Erdman Mtb strain knockout backgrounds, which aligns with 350 previous overexpression growth curve and relative growth data (Figure 2A  The stable viability of WT or ∆ppe51 mutant may be due to growth arrest or, alternatively, 358 balanced growth and death. To determine if the strains are truly growth arrested we examined 359 replication using a the pBP10 clock plasmid, transformed into WT Mtb, ∆ppe51, and eag variants 360 in both CDC1551 and Erdman backgrounds 53 . The strains were incubated in minimal media (pH 361 5.7 and 7.0) with glycerol for 40 adays. We observed that the WT and ∆ppe51 strains do not 362 replicate under acid growth arrest conditions in both strain backgrounds ( Figure 3C, Figure S10B). 363 In contrast, we are able to observe high rates of replication in the eag variants at pH 5.7 ( Figure  364 3C, Figure S10B). We then compared eag variants' calculated cumulative bacterial burden (CBB) 365 to total CFUs counted on nonselective plates and observed that greater rates of replication in the 366 eag variants is associated with a high death rate, yielding a large difference between CBB and total with all strains, we observed similar high rates of replication and plasmid loss across all strains 369 (Figure S10A and C). Interestingly, these results show that enhanced growth at acidic pH is driven 370 by higher replication, but this growth is offset somewhat by a higher death rate, supporting the 371 conclusion that faster replication at acidic pH is deleterious to Mtb survival. 372 373 Acidic pH limits glycerol uptake and PPE51 variants overcome this restriction. Pyruvate can 374 rescue growth on glycerol in a concentration-dependent manner at pH 5.7 18 . However, it is 375 unknown whether glycerol concentration affects acid growth arrest. We hypothesized that acid 376 growth arrest may be driven by glycerol starvation and the PPE51 variants promote growth by 377 promoting enhanced uptake of glycerol. If this is the case, we would expect to see a dependence 378 of glycerol concentration and acidic pH on growth. To examine this, we examined checker-board 379 dose responses combining varying pH levels (pH 6.5-5.5) and glycerol concentrations (80 mM-380 0.13 mM) using the panel of isogenic strains. The standard concentration of glycerol used in our 381 acid growth arrest model is 10 mM. Growth in the wells was analyzed using optical density (OD600) 382 and data was normalized to wells containing the highest (100%) levels of growth and wells with 383 no carbon representing the lowest (0%) levels of growth. Growth assays were performed for 21 384 days, and the data shown is Day 14 which is representative for the duration of the experiment. 385 Interestingly, we found that growth arrest appears to be both pH and glycerol concentration-386 dependent, with growth partially rescued at high concentrations of glycerol (~80 mM) for WT, 387 ∆ppe51::pMV306 and ∆ppe51::pMV-ppe51 at pH 5.7 (Figure 4 and S11A). Additionally, we 388 observed higher levels of growth at lower glycerol concentrations (~0.82 mM) at pH 5.7 with the 389 complemented ppe51 variants compared to the empty vector and WT ppe51 complemented strains.
Growth could also be rescued with high glycerol concentration (~32 mM) for variants at pH 5.5. 391 Interestingly, the presence of the double ppe51 variant (S211R+A228D) overcomes growth arrest 392 at pH 5.5 at even lower glycerol concentrations (~5.12 mM) compared to the single variants, 393 indicating that the presence of two eag point mutations confers a slight growth advantage during 394 acid growth arrest. Concentrations of glycerol below 0.33 mM do not rescue growth starting at pH 395 6.0 in any eag strains, which could be due to glycerol being fully consumed. Similarly, these 396 observations were also made in the native eag variants in both CDC1551 and Erdman, while WT 397 exhibited a reduced capacity for glycerol uptake ( Figure S11B). Together, these findings suggest 398 that Mtb has reduced capacity to uptake glycerol in a pH-dependent manner, and that PPE51 399 variants function by promoting enhanced uptake of glycerol. 400 Based on these checkerboard results, ppe51 appears to restrict its growth on glycerol at 401 acidic pH. Additionally, WT Mtb has been shown to completely arrest its growth at pH 5.7 on 10 402 mM glycerol; however, it is able to maintain viability for up to 40 days, remains metabolically 403 active, and incorporate limited amounts of exogenous 14 C-glycerol into lipids 16 . To further test the 404 hypothesis that Mtb restricts glycerol uptake at acidic pH and that eag variants promote enhanced 405 glycerol uptake, a radiolabeling experiment using 14 C-glycerol was conducted with WT Erdman 406 and the ∆ppe51 complemented strains previously described. Strains were pre-adapted for three 407 days in MMAT (pH 5.7 or 7.0) with 10 mM glycerol and washed with PBS prior to radiolabeling 408 with 6 µCi of 14 C-glycerol. Samples were collected over the course of 24 hours, washed, and 409 analyzed for radiolabel uptake by scintillation counting. All complemented strains containing a 410 ppe51 variant accumulated 14 C-glycerol at a similarly increased rate of approximately 300% 411 compared to the WT Mtb strain ( Figure 5A). These results are consistent with radiolabeling that 412 was conducted with the pVV16 overexpression empty vector, S211R overexpression strain, and native eag-S211R variant where we observed similar enhanced glycerol uptake at ~ 60% with 414 strains containing S211R compared to WT overexpression empty vector ( Figure S12A). We also 415 looked at glycerol uptake with WT CDC1551, empty vector, and complemented S211R at pH 7.0. 416 We did not observe significant differences in glycerol uptake between strains, and the rate of 417 uptake was similar to the complemented ppe51 variant strains at pH 5.7 ( Figure S12B). Together, 418 these results show that Mtb does restrict glycerol uptake at pH 5.7 regardless of whether ppe51 is 419 functionally intact. In contrast, strains containing ppe51 variants have significantly enhanced 420 glycerol uptake. 421 While the radiolabeling strongly indicated that glycerol was being taken up by the strains, 422 it did not answer whether glycerol was being metabolized by Mtb and incorporated into lipids or 423 binding to the mycomembrane without uptake across the plasma membrane. To address this 424 question, we performed lipid radiolabeling with 14 C-glycerol. WT Erdman and ∆ppe51 425 complemented strains were pre-adapted for three days in the same culture conditions as the 426 previously described radiolabeled uptake experiment. The operon controlling sulfolipid synthesis 427 is induced in a phoPR-dependent and a pH-dependent manner 18,50,56 . Sulfolipid was observed to 428 specifically accumulate at pH 5.7 ( Figure 5B) with no accumulation occurring at pH 7.0 (Figure 429 S12C). Triacylglycerol (TAG) has been shown to accumulate during periods of hypoxic and pH-430 stress 18,57 , and pathways involved in TAG synthesis play a role in reducing Mtb growth by 431 redirecting carbon flux away from the TCA cycle 58 . Interestingly, we found that TAG accumulated 432 specifically in the complemented S211R strain at pH 5.7 ( Figure 5C). In contrast, we did see 433 similar TAG accumulation across all strains at pH 7.0 ( Figure S12D). The observation of labeled 434 lipids in both growth arrested and growing Mtb at acidic pH, shows that glycerol is imported and 435 metabolized at acidic pH, with enhanced uptake in the S211R variant.

PPE51 variants have selectively reduced growth in activated macrophages. ppe51 is induced in 437
a pH-dependent and phoP-dependent manner within 2 hours following phagocytosis by 438 macrophages 10 , suggesting that ppe51 is important for pathogenesis. We hypothesized that ppe51 439 or its eag variants may be required for pathogenesis, specifically in activated macrophages, where 440 the phagosome is acidified. To test this hypothesis, resting and activated primary murine bone 441 marrow-derived macrophages (BMDMs) were infected with WT CDC1551 and ∆ppe51 mutant 442 and complemented variant strains. In resting macrophages, we did not observe significant 443 differences in Mtb growth between the strains ( Figure 6A Mtb killing by the macrophage 10 . We observed in vitro that variants had enhanced death during 451 replication at acidic pH, and we hypothesized that the eag variants may be replicating faster than 452 the WT in macrophages but have lower CFUs due to enhanced death rates. To test this hypothesis, 453 we infected BMDMs with native CDC1551 WT, Δppe51, and A228D variant containing the 454 pBP10 plasmid as described previously. Infection was conducted over the course of 8 days with 455 cells lysed and plated for viable CFUs every 2 days. We observed an initial ~0.5 log decrease in 456 viable CFUs in both WT and Δppe51 around day 2 that is consistent with observations made by 457 Rohde et. al. 59 , and supports their findings that Mtb exhibits delayed adaptation to survive and 458 replicate within macrophages ( Figure 6C). Both WT and Δppe51 then replicated over the course 459 of 8 days inside activated BMDMs as evident by their ~1 log increase in CFUs starting at day 2. 460 In contrast, the A228D variant lacks this initial adaptation period and instead show a continual ~1 461 log decrease in CFUs over the course of 8 days. Calculating the CBB of the A228D variant shows 462 a large difference between the CBB and CFUs, demonstrating that the A228D variant is replicating 463 at a higher rate and dying at an even greater rate. These strains are able to replicate and survive 464 better in resting BMDMs compared to activated BMDMs ( Figure S13). We conclude that slowed 465 growth in response to acidic pH inside activated macrophages is necessary for mycobacterial 466 survival and that the eag variants do not be sufficiently slow their growth inside macrophages, 467 resulting in enhanced killing. These results also support that the PPE51 variant is promoting uptake 468 of a carbon source during macrophage infection, suggesting that Mtb may metabolize glycerol 469 when growing in macrophages. 470 471

Differential thermal stability of PPE51 and the S211R variant proteins support direct 472
interactions between PPE51 and glycerol. We hypothesized that the eag variants promote PPE51 473 uptake of glycerol by altering PPE51 structure and its affinity for glycerol. Changes in the thermal 474 stability of the protein would provide evidence supporting this hypothesis. C-terminal his-tagged 475 recombinant PPE51 and PPE51 (S211R) variant proteins were expressed and purified from E. coli 476 ( Figure S14A and B, Table S1). Glycerol was omitted from the reagents used in the purification 477 process and loading dye. In the absence of glycerol, we observed differential stability between the 478 WT and S211R PPE51 variants, with the WT and S211R proteins completely denaturing at 60 ºC 479 and 50 ºC, respectively, supportive of a significant structural change by the amino acid substitution 480 ( Figure 7A and B). In glycerol, the WT protein exhibited enhanced stability, completely 481 denaturing at 65 ºC, a shift of 5 ºC, and the S211R protein completely denaturing at 60 ºC, a shift of 10 ºC. These findings support that glycerol/PPE51 interactions, with differential stability shifts 483 dependent on the S211R substitution. 484 We previously noted that PPE51 S211R did not promote the growth on glucose and therefore, 485 examined the thermal stability in glucose. We observed reduced stability of the WT protein in 486 glucose, completely denaturing at 55 ºC and did not observe any differences in stability with the 487 S211R protein in glucose, supporting the stability shifts are selectively dependent on glycerol 488 ( Figure 7A and B). Together, these data show that glycerol selectively increases the thermal 489 stability of PPE51, with enhanced impact on the S211R variant, lending further support for a 490 mechanism whereby PPE51 directly binds glycerol for uptake and acquisition into the Mtb cell. 491 Based on the eag phenotype and differences in thermal stability, we hypothesized that these 492 Furthermore, all eag mutations mapped to a single alpha helix on the predicted I-TASSER model, 510 with S211R and E215K located at the top of the predicted channel and A228D located within the 511 center channel structure ( Figure S14F) and the substitutions, altered the modeled substrate 512 interaction, further supporting our model for PPE51 variants acting to promote uptake of glycerol 513 by altering the protein structure and ligand interactions. 514 515 PDIM biosynthesis is disrupted in the ppe51 deletion strains. Surprisingly, we found that the 516 Δppe51 mutant generated in this study does not have the same growth or glycerol uptake compared 517 to previous studies that have generated similar knockouts of knockdowns of ppe51 28,41,61 . We also 518 found that the Δppe51 grew just as well as other strains at pH 7.0 on glycerol ( Figures 3B and  519   S8A). This observation was previously made by Wang et. al., who showed that mutations in 520 phthiocerol dimycocerosates (PDIM) biosynthesis were responsible for permeabilizing the 521 mycomembrane and compensating for the loss of functional ppe51 28 . We sequenced the genomes 522 of Δppe51 mutants in both the CDC1551 and Erdman backgrounds and found that both Δppe51 523 mutants had evolved mutations in PDIM biosynthesis pathway genes (ppsC in Δppe51-CDC1551, 524 and ppsD in Δppe51-Erdman). We confirmed for loss of functional PDIM by radiolabeling it with 525 14 C-glycerol and 14 C-acetate for six days and extracting total lipids for TLC analysis. As expected, 526 we observed loss of functional PDIM accumulation in the Δppe51::pMV-EV compared to WT Mtb 527 radiolabeled at both pH 5.7 and pH 7.0 with 14 C-acetate and 14 C-glycerol, respectively ( Figures 8B and S13). However, despite the occurrence of these PDIM mutations in the Δppe51 mutants, 529 no PDIM mutations were present in the sequenced eag variant mutants used in this study or WT 530 Mtb, highlighting that the eag variants remain gain-of-function, dominant mutants (Figure 2A), 531 that selectively promote uptake of glycerol ( Figure 2D) and promote enhanced uptake of glycerol 532 and enhanced replication in vitro and macrophages ( Figures 5A, 3C, 6C, S11B, S12A, S13). 533 Conclusions for the PPE51 knockout or eag variants in the ppe51 knockout background, must take 534 into account that these strains are also PDIM mutants. Overall, we did not observe any differences 535 in the eag mutants if they were in a WT or ppe51/PDIM mutant background, supporting that these 536 gain-of-function eag phenotypes are independent of PDIM levels. 537 538 Discussion 539 Mtb exhibits complex regulatory and physiological adaptations when grown in acidic 540 environments, including changes in growth rate. The underlying basis of slowed growth in mildly 541 acidic environments is still not fully resolved, but appears to be associated with metabolic and 542 redox stress, that may be linked to balancing cytoplasmic pH-homeostasis and respiration 11 . 543 Providing specific carbon sources, such as pyruvate or acetyl-CoA, relieve this metabolic stress 544 and enable Mtb to grow similarly well at acidic and neutral pH 16,18 . However, it has been puzzling 545 as to why Mtb cannot grow on glycerol at acidic pH, as it has a carbon source and oxygen, 546 everything it needs to grow. In this study, we found that Mtb limits uptake of glycerol at acidic pH 547 to restrict its growth and that mutations in ppe51 promote uptake of glycerol at acidic pH and 548 enable growth. That is, Mtb can grow well at acidic pH on glycerol, but has adapted instead to stop 549 growth. 550 We further show that this pH-dependent metabolic adaptation is required for pathogenesis. 551 Selectively in activated macrophages, where the pH of the phagosome is more acidic, we observed 552 a virulence defect in strains expressing the eag variants. Notably, using a replication clock plasmid, 553 we found that eag variants have enhanced growth in macrophages, but even greater killing, the 554 balance of which results in reduced fitness. Thus, slowed growth in macrophages, in an activation 555 dependent manner is dependent on the restriction of metabolism at acidic pH, and that PPE51 556 variants overcome this restriction to the detriment of the pathogen. This finding supports that the 557 nutrient imported by the PPE51 variant is relevant to the macrophage environment. We showed 558 that the variants specifically promote uptake of glycerol, therefore, it is plausible that glycerol is a 559 key regulator of Mtb growth in the macrophage. It has been previously shown that Mtb can uptake 560 TAG in macrophages 62 , TAG is abundant in granulomas 63 , and Mtb exports the TAG lipase 561 LipY 64 , therefore, it is possible glycerol is released from TAG during infection, and restriction of 562 glycerol uptake plays an important role in slowing growth during infection. Studies examining the 563 interactions of PPE51 eag variants, LipY and glycerol metabolism genes during pathogenesis will 564 be undertaken to test this hypothetical model. 565 It is a striking finding that all of the eag mutants selected were in ppe51 and that they all 566 clustered with a highly conserved region of 18 amino acid residues (residues 211-228). Three 567 single amino acid substitutions (S211R, A228D, and E215K) greatly altered WT ppe51 function 568 and promoted growth under acid stress when given the non-permissible carbon source, glycerol. 569 S211R was able to confer the greatest enhanced growth, whereas A228D conferred moderate 570 enhanced growth and E215K exhibited the least amount of enhanced growth, comparatively 571 ( Figure 2B). The growth phenotypes of the native mutant alleles were further recapitulated in 572 overexpression studies in a WT Mtb background as well as a Δppe51 background, where again we 573 observed overall greater eag with the S211R variant compared to A228D and E215K (Figures 2A,  574 3A, S8A). Given that the phenotype was conserved in PDIM containing strains (the initially 575 isolated mutants and the overexpressors in the WT) and PDIM lacking strains (the ppe51 deletion 576 mutants), this demonstrates that the gain-of-function phenotype is independent of PDIM. This 577 region of PPE51 may play a key role in protein-substrate interactions, and indeed with recombinant 578 proteins, we observed differential stability in the variant protein and its interaction with glycerol. 579 Interestingly, the structural modeling showed substitutions in this region altered the predicted 580 ligand of the modeled transporter, supporting further study of this critical region for modulating 581 PPE51-ligand interactions. 582 Another key finding of this study is that glycerol uptake is restricted at acidic pH. Data 583 supporting this conclusion include the reduced uptake of radiolabeled glycerol at acidic pH as 584 compared to neutral pH ( Figures 5A and S12AB), the dependence of glycerol concentration and 585 pH in regulating growth (Figures 4, S11A and S11B), and the ability of PPE51 variants to enhance 586 growth and glycerol uptake at acidic pH ( Figures 5A and S12AB). How Mtb restricts glycerol 587 uptake is still not known, but it is puzzling that PPE51 is strongly induced at acidic pH and counter 588 to a model where PPE51 promotes in glycerol uptake, but Mtb restricts glycerol uptake at acidic 589 pH. This contradiction remains unresolved and points to a new known unknown of Mtb 590 metabolism restriction at acidic pH. Notably, growth on glycerol-containing mixtures can exceed 591 growth compared to growth on glycerol alone 65 , suggesting that Mtb may need to restrict glycerol 592 to regulate its growth while consuming other carbon sources it encounters during infection. 593 We identified that the eag variants selectively enabled growth on glycerol alone compared 594 to WT Mtb ( Figure 2D). The identification of this carbon specificity with PPE51 eag variants 595 implies a putative role for PPE proteins in nutrient acquisition, a model that is strongly supported suggesting that PPE51 mediated impacts on carbon source uptake or mycomembrane permeability 617 play a role in drug susceptibility, supporting further studies of PPE51 as a target for potentiating 618 antibiotics.
Here, we present a model that integrates the current understanding of PE and PPE nutrient 620 acquisition with our findings (Figure 9), wherein PPE51 embeds itself into the outermost layer of 621 the cell envelope and is surface accessible to glycerol 28,42,73 . Gene expression profiling data 622 supports induction of ppe51 by phoP and acidic pH 15,18 . Phylogenetic evidence shows that PPE51 623 is duplicated alongside ESX-5 32 , which has been shown to mediate the secretion of most PE/PPE 624 proteins in M. marinum, including PPE51 34,38 . We propose that an unknown periplasmic nutrient 625 transporter helps mediate the import of glycerol across the plasma membrane and into the cell from 626 initial import by PPE51. pe/ppe families have high variation rates between Mycobacterium 627 tuberculosis complex (MTBC) genomes with ppe51 being the single exception in showing almost 628 no variation 74 . However, under the selective pressure of our screen ( Figure 1A), we have shown 629 that we can select for mutations that enhance PPE51's proposed uptake of glycerol (Figure 9). 630 Furthermore, our initial in silico modeling of PPE51 suggests that it can form a porin-like structure 631 consistent with a role in transport and ligand-binding sites for carbon nutrient sources ( Figure 8C). 632 Based on these data, we further propose an eag mutant model, whereby the eag amino acid 633 substitutions introduce conformational changes that allow for a possible PPE51-porin structure to 634 widen or enhance the binding the glycerol, allowing enhanced transport through the 635 mycomembrane. 636 This study has focused on the role of the eag PPE51 variants, and the not the Dppe51 mutant, 637 due to confounding mutations in PDIM in the deletion strains. It is interesting that both deletion 638 mutants (in Erdman and CDC1551) evolved these mutations during the construction of the mutants 639 and suggests there may have been a selective advantage for the mutations. Indeed, Wang et al., 640 showed that ppe51 knockouts only had a glycerol uptake phenotype when the PDIM was restored 641 in the mutant. This finding is consistent with our observation that the Dppe51 mutants in this study did not have a growth defect in glycerol, presumably due to the lack of PDIM, whereas the ppe51 643 mutants in the Wang et al., study were defective for growth. Given the conservation of the eag 644 mutants in strains with or without PDIM, we conclude that PDIM level do not appreciably impact 645 the enhanced uptake of glycerol in eag variants. However, it is also possible that differences for 646 the PPE51 mutants between this study and the others may be driven by genomic differences. Both

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and Δppe51 complemented strains in minimal media supplemented in a dose-dependent manner with 715 glycerol and buffered to one of five pH levels (pH 6.5, 6.2, 6.0, 5.7, or 5.5). All strains exhibit a reduced capacity for growth starting ~2 mM glycerol compared to higher glycerol concentrations. At decreasing 717 pH, WT, Δppe51(empty vector), and Δppe51::pMV-WT restrict their ability to uptake glycerol, whereas 718 any variant complement is able to maintain glycerol uptake. However, restricted growth can be rescued at 719 high concentrations of glycerol (~80 mM) at pH 5.7 for WT, Δppe51(empty vector), and Δppe51::pMV-720 ppe51, and pH 5.5 for variant complements. Growth analyses were performed at Day 14 following initial 721 inoculation with data being shown as percent of the maximum well-growth. All conditions were conducted                      with glycerol and compared to the WT for the enhanced acid growth phenotype. Each symbol represents the numbered colony isolated from the acid growth arrest plates.